Biodegradable targetable microparticle delivery system

ABSTRACT

Copolymers designed for use as particulate carriers containing functionalizable amino acid subunits for coupling with targeting ligands are described. The copolymers are polyesters composed of α-hydroxy acid subunits such as D,L-lactide and pseudo-α-amino acid subunits which may be derived from serine or terpolymers of D,L-lactide and glycolide and pseudo-α-amino acid subunits which may be derived from serine. Stable vaccine preparations useful as delayed release formulations containing antigen or antigens and adjuvants encapsulated within or physically mixed with polymeric mircoparticles are described. The particulate carriers are useful for delivering agents to the immune system of a subject by mucosal or parenteral routes to produce immune responses, including antibody and protective responses.

REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending U.S. Pat. Ser.No. 08/770,050 filed Dec. 20, 1996 now U.S. Pat. No. 6,042,820.

REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. 371 ofPCT/CA97/00980 filed Dec. 19, 1997, which is a continuation-in-part ofU.S. patent application Ser. No. 08/770,850 filed Dec. 20, 1996 (nowU.S. Pat. No. 6,082,820).

FIELD OF THE INVENTION

The present invention relates to biodegradable microparticles fordelivery of a biologically active material and is particularly concernedwith such microparticles that are targetable to particular cell types.

BACKGROUND OF THE INVENTION

Vaccines have been used for many years to protect humans and animalsagainst a wide variety of infectious diseases. Such conventionalvaccines consist of attenuated pathogens (for example, polio virus),killed pathogens (for example, Bordetella pertussis) or immunogeniccomponents of the pathogen (for example, diphtheria toxoid and hepatitisB surface antigen).

Some antigens are highly immunogenic and are capable alone of elicitingprotective immune responses. Other antigens, however, fail to induce aprotective immune response or induce only a weak immune response. Theimmune response of a weakly immunogenic antigen can be significantlyenhanced if the antigens are co-administered with adjuvants. Adjuvantsenhance the immunogenicity of an antigen but are not necessarilyimmunogenic themselves. Adjuvants may act by retaining the antigenlocally near the site of administration to produce a depot effectfacilitating a slow, sustained release of antigen to cells of the immunesystem. Adjuvants can also attract cells of the immune system to anantigen depot and stimulate such cells to elicit immune responses.Adjuvants have been identified that enhance the immune response toantigens delivered parenterally.

Adjuvants are commonly employed with antigen in vaccine formulationswhereby the induction of systemic immunity through parenteralimmunization (intramuscular or subcutaneous) is obtained. This approachis suitable for infectious agents gaining access to the body via damagedskin (i.e. Tetanus), however, there are problems encountered due toside-effects and associated toxicity of many adjuvants administered inthis fashion. Only those vaccines formulated from aluminum salts(aluminum phosphate or aluminum hydroxide) find routine use in human andveterinary vaccination. However, even these adjuvants are not suitablefor use with all antigens and can also cause irritation at the site ofinjection. There is a clear need to develop adjuvants which safelyenhance the immunogenicity of antigens at the site of injection.

There are other problems specific to the nature of the antigen beingused. For example, most conventional non-living vaccines requiremultiple doses for effective immunization. Live attenuated vaccines andmany nonliving liquid vaccines suffer from the need for controlledstorage conditions and are susceptible to inactivation (e.g. thermalsensitivity). There are also problems associated with combining vaccinesin single dosage forms, due to adjuvant incompatibilities, pH, buffertype and the presence of salts.

Mucosal immunity is induced primarily by induction of secretoryimmunoglobulin (sIgA) in intestinal, bronchial or nasal washings andother external secretions. For example, parenteral cholera vaccines havebeen shown to offer limited protection whereas the more recentlydeveloped oral form is highly effective (ref. 1—throughout thisspecification, various references are referred to in parenthesis to morefully describe the state of the art to which this invention pertains.Full bibliographic information for each citation is found at the end ofthe specification, immediately preceding the claims. The disclosures ofthese references are hereby incorporated by reference into the presentdisclosure). Studies with human volunteers have shown that oraladministration of influenza vaccine is effective at inducing secretoryanti-influenza antibodies in nasal secretions and substances have beenidentified which might be useful as adjuvants for such ingestedvaccines. However, most of these adjuvants are relatively poor in termsof improving immune responses to ingested antigens. Currently, most ofthese adjuvants have been determined to be safe and efficacious inenhancing immune responses in humans and animals to antigens that areadministered via the orogastrointestinal, nasopharyngeal-respiratory andgenital tracts or in the ocular orbits. However, administration ofantigens via these routes is generally ineffective in eliciting animmune response. Although the above example illustrates the potential ofthese immunization modes, the development of vaccine formulations foruse by these routes has been slow for various reasons. The inability toimmunize at the mucosal surface is generally believed to be due toinclude:

(i) antigen degradation via the acid and/or proteolytic enzymes presentduring the transit to the mucosal surfaces;

(ii) antigen degradation by secretions presented at the mucosalepithelium;

(iii) limited adsorption across the mucosal epithelium;

(iv) the dilution of the antigen to a concentration that is below thatrequired to induce immune responses; and

(v) ineffective adjuvants and/or delivery systems.

The problems associated with the use of adjuvants in parenteral vaccineformulations and the lack of suitable systems for vaccine delivery tomucosal sites understates the need for new techniques that are effectivewhen administered by various routes and are inherently free fromassociated toxicity concerns or side-effects.

It is also desired to provide vaccine delivery in a single dosage formfor both human and animal immunizations as this has the advantage ofreducing time and cost, and in human medicine, increases patientcompliance which is of extreme importance in developing countries whereaccess is restricted. This is especially true for infants within thesecountries.

In order to increase immune responses to administered antigens, acarrier may be used to protect the antigen from degradation and alsomodulate the uptake of these materials in vivo. Sensitive antigens maybe entrapped to protect them against destruction, reduction inimmunogenicity or dilution. Methods for formulating a carrier includedispersing an antigen within a polymeric matrix (monolithic matrix) orby the coating of a polymeric material around an antigen to give anouter protective wall (core-shell). The manipulation of the formulationprotocol can allow for control over the average size of these materials.This has been shown to be important for the uptake of particulates viaoral delivery at specialized M-cells of the Peyers patches within theintestinal tract.

U.S. Pat. No. 5,151,264 describes a particulate carrier of aphospholipid/glycolipid/polysaccharide nature that has been termed BioVecteurs Supra Moleculairs (BVSM). The particulate carriers are intendedto transport a variety of molecules having biological activity in one ofthe layers thereof. However, U.S. Pat. No. 5,151,264 does not describeparticulate carriers containing antigens for immunization andparticularly does not describe particulate carriers for immunization viathe orogastrointestinal, nasapharyngeal-respiratory and urogenitaltracts and in the ocular orbits or other mucosal sites.

Eldridge et al.(refs 2 and 3) observed the delayed release of antigen invivo from biodegradable microspheres manufactured frompolylactide-co-glycolide copolymer also known as PLG or PLGA. Numerousother polymers have been used to encapsulate antigens for formulationinto microparticles and some of these include polyglycolide,polylactide, polycaprolactone, polyanhydrides, polyorthoesters andpoly(α-hydroxybutyric acid).

U.S. Pat. No. 5,075,109 describes encapsulation of the antigenstrinitrophenylated keyhole limpet hemocyanin and staphylococcalenterotoxin B in 50:50 poly (DL-lactide-co-glycolide). Other polymersfor encapsulation are suggested, such as poly(glycolide),poly(DL-lactide-co-glycolide), copolyoxalates, polycaprolactone,poly(lactide-co-caprolactone), poly(esteramides), polyorthoesters andpoly(α-hydroxybutyric acid), and poly anhydrides. The encapsulatedantigen was administered to mice via gastric intubation and resulted inthe appearance of significant antigen-specific IgA antibodies in salivaand gut secretions and in sera. As is stated in this patent, incontrast, the oral administration of the same amount of unencapsulatedantigen was ineffective at inducing specific antibodies of any isotypein any of the fluids tested. Poly(DL-lactide-co-glycolide) microcapsuleswere also used to administer antigen by parenteral injection.

Published PCT application WO 91/06282 describes a delivery vehiclecomprising a plurality of bioadhesive microspheres and antigenic vaccineingredients. The microspheres being of starch, gelatin, dextran,collagen or albumin. This delivery vehicle is particularly intended forthe uptake of vaccine across the nasal mucosa. The delivery vehicle mayadditionally contain an absorption enhancer. The antigens are typicallyencapsulated within protective polymeric materials.

U.S. Pat. No. 5,571,531 describes particulate carriers comprising asolid matrix of a polysaccharide and a proteinaceous material. Afunctionalized silicone polymer is bonded to the matrix for the deliveryof materials having biological activity.

Although time-delayed release of antigen was shown in the above work,difficulties were encountered when microparticles are manufactured bythe described methods. The exposure of biological materials to theorganic solvents and physical forces used can lead to denaturation. Itmay be also be difficult to scale-up the procedures. Furthermore,hydrophilic antigens may be inefficiently encapsulated.

It would be desirable to provide improved carriers without suchlimitations. It would be particularly desirable to provide polymericmaterials which can be formulated into microparticles and microspheresand which contain targeting moieties to target the antigen topreselected ligands. This would have tremendous potential for cells ofthe immune system.

SUMMARY OF THE INVENTION

The present invention is directed towards the production of a novel anduseful polymer that has properties suitable for manufacturing by variousprocesses into microparticles and microspheres. In this invention,modifications of existing processing procedures results in significantimprovement in encapsulation efficiencies.

This invention is further directed to the production of useful vaccinedelivery systems for antigen(s) or antigen and co-adjuvant cocktails byvarious immunization routes which include parenteral, oral andintranasal.

In accordance with a first aspect of the invention, there is provided anovel biodegradable, biocompatible polymer, including, those having amolecular weight of about 5,000 to about 40,000 daltons, having abackbone of the general formula:

wherein;

R₁, R₂, R₃, R₄ and R₅ are selected independently and are selected fromH, linear or branched alkyl groups;

R₆ is selected from H, an amine protecting group, a spacer molecule or abiologically active species;

X is selected from an O or S group; and

x and y are integers, including values such that at least about 95% ofthe polymer is comprised of α-hydroxy acid residues.

The novel polymers are derived by copolymerization of monomerscomprising at least one α-hydroxy acid or derivative thereof, includingcyclic divesters and at least one pseudo-α-amino acid. The α-hydroxyacids are generally of the formula R₁R₂COHCO₂H, where the R₁ and R₂groups are H, linear or branched alkyl groups. The α-hydroxy acids maycomprise a mixture of α-hydroxy acids, at least one of the mixture ofα-hydroxy acids having R₁ and R₂ groups which are hydrogen and anotherα-hydroxy acid having an R₁ group which is CH₃ and R₂ which is H. Thepseudo-α-amino acids are generally of the formula R₅CHNHR₆CO₂H, wherethe R₅ group is a hydroxyl methyl or methyl thiol group and R₆ is anamine protecting group.

The amine protecting groups may be carbobenzyloxy, benzyl,paramethoxybenzyl, benzyloxymethoxy, tert-butyloxycarbonyl or[9-fluorenylmethyloxy]carbonyl.

The α-hydroxy acids are generally selected from L-lactic acid,D,L-lactic acid, glycolic acid, hydroxy valeric acid and hydroxybutyricacid. The at least one pseudo-α-amino acid may be serine.

In a preferred aspect of the invention, the polymers arepoly-D,L-lactide-co-glycolide-co-pseudo-Z-serine ester (PLGpZS) andpoly-P,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS).

The polymers may contain biologically active moieties, such as cellbioadhesion groups, macrophage stimulators, polyethylene glycol, polyamino acids and/or protected amino acid residues, covalently bound tothe polymer directly or through side groups.

In the preferred embodiment, the bioactive substituents are linked tothe polymer via the amino groups on the amino acid moieties directly orvia a suitable spacer molecule. The spacer molecule can be selected fromα-hydroxy acids represented by the formula R₇R₈COHCO2H, where R₇ or R₈groups are independently selected from H, linear or branched alkyl unitsand α-amino acids represented by the formula R₉CHNHR₁₀CO₂H, where the R₉group is a hydroxyl methyl or methyl thiol group and R₁₀ is an amineprotecting group.

In accordance with a further aspect of the invention, the inventionprovides a method of making a biodegradable, biocompatible polyester,which comprises co-polymerizing at least one α-hydroxy acid and at leastone pseudo-α-amino acid.

In accordance with another aspect of the present invention, there isprovided a process for making a biodegradable, biocompatible polymer ofthe general formula provided herein which comprises forming a mixture ofmonomers comprising at least one α-hydroxy acid and at least onepseudo-α-amino acid with an organic solvent solution of anesterification catalyst under inert atmospheric conditions,copolymerizing the monomers and isolating the resultant polymer. Thecatalyst used is preferably stannous 2-ethylhexanoate.

The polymer formed by the process can be further deprotected by solidphase catalytic reduction or alternatively by acid catalysis usinghydrogen bromide in acetic acid solution.

The process can also further comprise forming the polymer into a film ormicroparticles.

In accordance with another aspect of this invention, there is provided aparticulate carrier for the delivery of biologically active materials toa host, the carrier comprising a polymer, including those having amolecular weight of about 5,000 to about 40,000 daltons, having thegeneral formula:

wherein;

R₁, R₂, R₃, R₄ and R₅ are selected independently and are selected fromH, linear or branched alkyl groups;

R₆ is selected from H, an amine protecting group, a spacer molecule or abiologically active species;

X is selected from an O or S group; and

x and y are integers, including values such that at least about 95% ofthe polymer is comprised of α-hydroxy acid residues.

The particulate carrier generally has a particle size of about 1 to 50μM.

In a further aspect of the present invention is a process for making aparticulate carrier for the delivery of at least one biologically activematerial to a host, the process comprising:

(a) mixing an organic solvent phase comprising an α-hydroxy acid polymeror copolymer with an aqueous composition comprising dispersed ordissolved biologically active material to form a first water-in-oilemulsion;

(b) dispersing the first water-in-oil emulsion into an aqueous detergentphase to form a second water-in-oil-in-water double emulsion;

(C) removing water from the second double emulsion to form microspheres;and

(d) collecting the microspheres and having the biological materialentrapped therein.

The particulate carrier of the present invention can be used as acomposition having a biologically active material mixed therewith orentrapped within. The biological materials used may be selected fromthose which elicit an immune response. Such materials may compriseHaemophilus influenzae proteins, such as a non-proteolytic Hin-47analog, D15, P1, P2, and P6. The biologically-active material maycomprise at least one influenza virus, which may be a multivalent ormonovalent influenza virus vaccine, or influenza virus protein, such asan influenza virus monovalent protein vaccine. In addition, thebiologically-active material may comprise at least one Moraxellacatarrhalis protein, such as the Tbp2 protein of M. catarrhalis. Afurther biologically-active material which may be employed may be atleast one Helicobacter pylori protein, such as Urease. Other biologicalmaterial may include proteins, protein mimetics, bacteria, bacteriallysates, viruses (e.g. respiratory syncytial virus), virus-infected celllysates, DNA plasmids, antisense RNA, peptides (e.g. CLTB-36 and M2),antigens, antibodies, pharmacological agents, antibiotics,carbohydrates, lipids, lipidated amino acids (e.g. tripalmitoylcysteine), glycolipids, haptens and combinations and mixtures thereof.

The first water-in-oil emulsion may additionally comprise at least oneorganic solvent soluble adjuvant, which may be lipophilic. Such organicsolvent adjuvant may be selected from the group consisting of BAYR1-005, tripalmitoyl cysteine and DC-chol. The presence of a lipophilicmoiety serves to increase the encapsulation efficiency and to protectthe antigen during formulation and release and enables the particles topresent antigen to the immune system more efficiently than traditionalformulation and hence provides a more efficacious vaccine.

The first water-in-oil emulsion also may additionally comprise at leastone water soluble adjuvant, which may be a polymeric water solubleadjuvant, such as PCPP or a mucosal adjuvant, such as CT-X or subunitthereof or LT. The presence of the water soluble adjuvant serves toincrease the encapsulation efficiency of the process and protects theantigen during formulation and release and prevents the antigen to theimmune system more efficiently than traditional microparticleformulation, thereby providing a more efficancious vaccine.

The present invention also provides an immunogenic compositioncomprising the particulate carrier provided herein and a physiologicallyacceptable carrier therefor. The composition can be administeredmucosally or parenterally. The immune response is an antibody responsewhich is a local or serum antibody response. In accordance with thisaspect of the invention, there is provided a controlled or delayedrelease vaccine preparation in stable particulate form and a method ofmaking such a vaccine preparation. The particles are microspherical andcontain a matrix of biodegradable polymer and antigen(s) and/or antigenplus co-adjuvant containing regions.

Advantages of the invention include:

(a) fully biodegradable and biocompatible microparticle formulation;

(b) facilitated antigen presentation to the cells of the immune systemresulting in improved antigen immunogenicity;

(c) improved formulating conditions which increase the bioavailabilityof the antigen.

Additional embodiments of the present invention include the use of theparticulate carrier in diagnostic assays and for therapeutic strategies.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the followingdescription with reference to the drawings, in which:

FIG. 1 is a schematic showing the ring opening polymerization (ROP) oflactic acid dimer with glycolic acid dimer andN-(carbobenzyloxy)-L-serine lactone with subsequent deprotection inaccordance with a preferred aspect of the present invention.

FIG. 2 is a schematic showing the attachment of biologically activemoieties to polymer through the side chain of the a-amino acid sub-unitwithin the polymer. Representative targeting groups includepoly-ethylene glycol (PEG) for water solubility and circulation,macrophage stimulators and cell bioadhesion groups. Spacer ligandsderived from α-hydroxy acids or α-amino acids may be incorporated tofacilitate attachment of the bioactive ligand.

FIG. 3 is a schematic detailing the process used to producemicroparticles in accordance with one embodiment of the invention. Inthis figure, Hin-47 is a non-proteolytic recombinant protein analogderived from Haemophilus influenzae (as described in U.S. Pat. No.5,506,139), Flu X31 is influenza strain X31 or A-Texas, rD-15 isrecombinant protein derived from Haemophilus influenzae (as described inWO 94/12641), PVA=poly vinyl alcohol. Flu(tri) is trivalent flu, Tbp2 isMoraxella catarrhalis transferrin binding protein 2 and rUrease isrecombinant Helicobacter pylori urease.

FIG. 4 shows a typical size distribution forpoly-D,L-lactide-co-glycolide-co-pseudo-(Z)-serine ester (PLGpZS) andpoly-D,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS)microparticles when prepared in the presence of PBS or a typical protein(non-proteolytic Hin-47 analog) as determined by laser diffractionmeasurements.

FIG. 5 shows a scanning electron micrograph of microparticles preparedfrom poly-D,L-lactide-co-glycolide-co-pseudo-(Z)-serine ester (PLGpZS)and poly-D,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS) in thepresence of phosphate buffered saline(PBS).

FIG. 6 shows a scanning electron micrograph of microparticles preparedfrom poly-D,L-lactide-co-glycolide-co-pseudo-(Z)-serine ester (PLGpZS)and poly-D,L-lactide-co-glycolide-co-pseudo-serine ester (PLGpS) in thepresence of a typical antigen/PBS mixture such as Hin-47/PBS.

FIG. 7A shows the in vitro release profile for non-proteolytic Hin-47analog encapsulated within PLG, PLGpZS and PLGpS microparticles over athree month period obtained from 14 mg samples (typical core loadingsrange from 2.5 to 5.7 μg protein/mg of microparticles) that wereincubated in PBS (pH=7.4) and maintained at 37° C.

FIG. 7B compares the % cumulative release of non-proteolytic Hin-47analog from PLG, PLGpZS and PLGpS microparticles over a three monthperiod obtained from ˜14 mg samples (typical core loadings range from2.5 to 5.7 μg protein/mg of microparticles) that were incubated in PBS(pH=7.4) at maintained and 37° C.

FIG. 8A shows the serum IgG responses in mice immunized subcutaneously(S.C.) following various immunization protocols by the 47 kDa membraneprotein from Haemophilus influenzae (non-proteolytic Hin-47 analog).Groups of 5 mice were immunized on days 1 and 35 with 250 μL of PBS, pH7.4, containing either 0.2 or 0.6 μg of non-proteolytic Hin-47 analogincorporated into PLG, PLGpZS or PLGpS microparticles. Sera obtained ondays +10, +24, +35, +46 and +60 were evaluated for the presence ofanti-Hin-47 IgG antibodies using an enzyme-linked immunosorbent assay(ELISA).

FIG. 8B shows the serum IgG responses in mice immunized subcutaneously(S.C.) following various immunization protocols by the 47 kDa membraneprotein from Haemophilus influenzae (non-proteolytic Hin-47 analog).Groups of 5 mice were immunized on days 1 and 35 with 250 μL of PBS, pH7.4, containing either 0.8 or 2.5 μg of non-proteolytic Hin-47 analogphysically mixed with PLG microparticles. Sera obtained on days +10,+24, +35, +46 and +60 were evaluated for the presence of anti-Hin-47 IgGantibodies using an enzyme-linked immunosorbent assay (ELISA).

FIG. 8C shows the serum IgG response subtype profile for pooled bleedsobtained on days +35 and +60 from the study conducted as described inFIG. 8A and 8B.

FIG. 9A shows the IgG serum antibody responses in mice immunizedintragastrically (I.G.) by the 47 kDa membrane protein from Haemophilusinfluenzae (non-proteolytic Hin-47 analog). Groups of 5 mice wereimmunized on days 1, 7, 14 and 57 with 500 μL of PBS, pH 7.4, containing4 pg of Hin-47 analog incorporated into PLG, PLGpZS or PLGpSmicroparticles or physically mixed with PLG, PLGpZS or PLGpSmicroparticles. Sera obtained on days +13, +35, +56 and +78 wereevaluated for the presence of anti-Hin-47 IgG antibodies using anenzyme-linked immunosorbent assay (ELISA).

FIG. 9B shows the serum IgG response subtype profile for pooled bleedsobtained on days +56 and +78 from the study conducted as illustrated inFIG. 9A.

FIG. 9C shows the serum IgA response for the bleed obtained on day +78from the study conducted as illustrated in FIG. 9A.

FIG. 10A shows the IgG serum antibody responses in mice immunizedintranasally (I.N.) by a (1:1) cocktail of the 47 kDa membrane proteinfrom Haemophilus influenzae (non-proteolytic Hin-47 analog) and the 115kDa membrane protein from Haemophilus influenzae (rD-15). Groups of 5mice were immunized on days 1, 7, 14 and 57 with 25 μL of PBS, pH 7.4,containing 4 μg of non-proteolytic Hin-47 analog incorporated into PLG,PLGpZS or PLGpS microparticles or physically mixed with PLG, PLGpZS orPLGpS microparticles. Sera obtained on days +13, +35, +56 and +78 wereevaluated for the presence of anti-Hin-47 IgG antibodies using anenzyme-linked immunosorbent assay (ELISA).

FIG. 10B shows the serum IgG response subtype profile for pooled bleedsobtained on days +56 and +78 from the study conducted as described inFIG. 10A.

FIG. 10C shows the serum IgA response for the bleed obtained on day +78from the study conducted as described in FIG. 10A.

FIG. 10D shows the lung lavage IgG response obtained on day +78 from thestudy conducted as described in FIG. 10A.

FIG. 10E shows the lung lavage sIgA response obtained on day +78 fromthe study conducted as described in FIG. 10A.

FIG. 11 shows the anti-Flu X31 (i.e. influenza virus type A strain X31)IgG serum antibody responses following various immunization protocols.Groups of 6 mice were immunized subcutaneously (S.C.) on day 1 with 250μL of PBS, pH 7.4, containing 1.5 μg of HA incorporated into PLG, PLGpZSor PLGpS microparticles. Sera obtained on days +21 and +33 and wereevaluated for the presence of anti-Flu X-31 IgG antibodies using anenzyme-linked immunosorbent assay (ELISA).

FIG. 12 shows the anti-Flu X31 (i.e. influenza virus type A strain X31)IgG serum antibody responses following various immunization protocols.Groups of 6 mice were immunized intranasally (I.N.) on days 1 and 34with 25 μL of PBS, pH 7.4, containing 1.5 μg of HA incorporated intoPLG, PLGpZS or PLGpS microparticles. Sera obtained on days +21, +33,+57, +78 and +92 were evaluated for the presence of anti-Flu X-31 IgGantibodies using an enzyme-linked immunosorbent assay (ELISA).

FIG. 13 shows the hemagglutination inhibition antibody assay (i.e.influenza virus strain A-Texas) responses for pooled sera (days +21 and+42 or +57) following a single dose subcutaneous administration. Groupsof 6 mice were immunized subcutaneously (S.C.) On day 1 with 250 μl ofPBS, pH 7.4, containing either 0.35 μg of HA or 3.5 μg of HAincorporated into PLGpS microparticles or 0.35 μg of HA and ˜2 μg of BAYR1-005 or 3.5 μg of HA and ˜20 μg of BAY R1-005 incorporated into PLGpSmicroparticles or 0.35 μg of HA or 3.5 μg of HA physically mixed withPLGpS microparticles or 0.35 μg of HA and 2 μg of BAY R1-005 or 3.5 μgof HA and 20 μg of BAY R1-005 physically mixed with PLGpSmicroparticles. Sera obtained on days +21 and +42 were evaluated for theinhibition of hemagglutination of erythrocytes.

FIGS. 14a to c show the anti-Flu (trivalent) IgG serum antibodyresponses (for each influenza virus strain contained in trivalentvaccine; A/Texas (FIGS. 14a), A/Johannesburg (FIG. 14b) and B/HarbinFIG. 14C)) following various immunization protocols. Groups of 8 micewere immunized subcutaneously (S.C.) on day 1 with 250 μL of PBS, pH7.4, containing 2.35 μg of total HA (about ⅓ specific HA from eachstrain) incorporated into PLGpS microparticles or PLGpS microparticlesformulated in the presence of BAY R1-005, DC-Chol or PCPP. Sera obtainedon days +21, +42 and +57 and were evaluated for the presence of anti-Flu(trivalent) IgG antibodies (A/Texas, A/Johannesburg and B/Harbin) usingan enzyme-linked immunosorbent assay (ELISA).

FIGS. 15a to c show the strain specific hemagglutination inhibitionantibody assay (i.e. A/Texas (FIG. 15a), A/Johannesburg (FIG. 15b) andB/Harbin (FIG. 15c)) responses (days +21, +42 and +57) following asingle dose subcutaneous administration. Groups of 8 mice were immunizedsubcutaneously (S.C.) on day 1 with 250 μL of PBS, pH 7.4, containingeither 2.35 μg of total HA incorporated into PLGpS microparticles or2.35 μg of total HA into PLGpS microparticles coencapsulating BAYR1-005, DC-Chol or PCPP. Sera obtained on days +21, +42 or +57 wereevaluated for the inhibition of hemagglutination of erythrocytes.

FIG. 16 shows the results of a protection study performed on miceimmunized with a single dose of PLGpS/Flu (trivalent) microparticles orPLGpS/Flu (trivalent) coencapsulating BAY R1-005, DC-Chol or PCPP. Themice were challenged with homologous live virus and monitored for weightchanges and survival over a 2 week interval.

FIG. 17a shows the serum IgG antibody responses in mice immunizedsubcutaneously (S.C.) following various immunization protocols by thetransferrin binding protein from Moraxella catarrhallis (Tbp-2) Groupsof 5 mice were immunized on days 1, 28 and 43 with 250 μL of PBS pH 7.4,containing 0.3 μg of tbp-2 incorporated into PLGpS microparticles,physically mixed PLGpS microparticles or formulated with Alum. Seraobtained on days +14, +27, +42 and +55 were evaluated for the presenceof anti-tbp-2 IgG antibodies using an enzyme-linked immunosorbent assay(ELISA).

FIG. 17b shows the serum IgG antibody subtype response profile forpooled bleeds obtained on day 55 from the study conducted as describedin FIG. 17a.

FIG. 18 shows the IgG serum antibody responses in mice immunizedintranasally (I.N.) with the transferrin binding protein from Moraxellacatarrhallis (Tbp-2). Groups of 5 mice were immunized on days 1, 28 and43 with either 10 μL or 50 μL of PBS, pH 7.4, containing 6 μg of Tbp-2incorporated into microparticles or physically mixed with PLGpSmicroparticles. Sera obtained on days +14, +27, +42 and +55 wereevaluated for the presence of anti-Tbp-2 IgG antibodies using anenzyme-linked immunosorbent assay (ELISA).

FIG. 19 shows the serum IgG antibody responses in guinea pigs immunizedparenterally following various immunization protocols by the transferrinbinding protein from Moraxella catarrhallis (Tbp-2). Groups of 2 guineapigs were immunized intramuscularly with 5 μg of Tbp-2 formulated with400 μL of CFA on day 1 followed subcutaneously with 5 μg of Tbp-2formulated in 500 μL of IFA on days 14 and 28, 5 μg of Tbp-2 formulatedwith 500 μL of Alum on days 1, 14 and 28 or 5.0 μg of Tbp-2 incorporatedinto PLGpS microparticles on days 1 and 28. Sera obtained on days +40and +56 were evaluated for the presence of anti-tbp-2 IgG antibodiesusing an enzyme-linked immunosorbent assay (ELISA).

FIG. 20 shows the serum IgG antibody subtype responses in mice immunizedsubcutaneously (S.C.) or orally (I.G.) following various immunizationprotocols by the recombinant protein rUrease from Helicobacter pylori.Groups of 8 mice were immunized on days 1, 28 and 56 with 250 μL of PBSpH 7.4, containing 10.0 μg (S.C.) or 40.0 μg (I.G.) of rUreaseincorporated into PLGpS microparticles or 10.0 μg (S.C.) or 40.0 μg(I.G.) of rUrease formulated in the presence of DC-Chol, CT-X, PCPP orLT incorporated into PLGpS microparticles. Sera was obtained on day +85and were evaluated for the presence of anti-rUrease IgG antibodies usingan enzyme-linked immunosorbent assay (ELISA).

FIGS. 21a to b show the results of a protection study for the micedescribed in FIG. 20 one month after challenge on day 85. rUreaseactivity (for the mice immunized by subcutaneous or oral routes) wasmeasured in ¼ of a whole stomach (antrum+corpus) 24 hours after the micewere killed.

DETAILED DESCRIPTION OF THE INVENTION

The novel polymers of the present invention are biocompatible,degradable to benign metabolites which may be present in the body andmay possess biologically active moieties, such as cell bioadhesiongroups, macrophage stimulators, poly amino acids and polyethylene glycolcoupled to the polymer via at least one spacer molecule selected fromα-hydroxy acids and α-amino acids. As such, the novel polymers possessfunctionality.

Methods are also described for the synthesis of polymers havingadvantageous properties for processing into microparticles containingbiologically active materials and for which chemical modification withbiologically active targeting groups is possible.

In the preferred embodiments, the copolymers are produced by thepolymerization of α-hydroxy acids with pseudo-α-amino acids andterpolymers produced by the polymerization of two α-hydroxy acids withpseudo-α-amino acids. The copolymer or terpolymer may then bederivatised with biologically active targeting ligands via the aminoacid subunit by covalently coupling with the free amino group directlyor subsequent to further derivatization with a suitable spacer ligand.

Amino Acid Monomer Synthesis

In general, an N-protected serine (or cysteine) is cyclized via aMitsunobu reaction (ref. 5) to give a four membered lactone (orthiolactone).

This transformation gives rise to an ester (or thioester) linkage. It isimportant to have protection on the amine portion of the amino acidprecursor that is compatible with the reaction conditions.Preferentially the carbobenzyloxy (CBZ or Z) group is used althoughother suitable functionalities, such as benzyl (Bn), para-methoxybenzyl(MeOBn), benzyloxymethoxy (BOM), tert-butyloxycarbonyl (t-BOC) or[9-fluorenylmethyl) oxy]carbonyl (FMOC) may be employed.

The synthesis of the N-Z-L-Serine β-Lactone monomer was based on amodified procedure from the literature (ref. 6).

Copolymerization Of α-Hydroxy Acid And Amino Acid Containing Monomersand Functionalization of Amino Acid Sidechains

Two methods are applicable for copolymerization of α-hydroxy acidmonomers. Polymerization via polycondensation or from the melt (bulkpolymerization) are possible alternatives.

It has been long known that condensation polymerizations are problematicas relatively low molecular weight materials often result with competingside reactions commonly giving rise to unwanted byproducts (refs. 7 and8).

However ring opening polymerization (ROP) of the cyclic dimers ofα-hydroxy acids, such as glycolide and lactide, from the bulk phase wasshown to proceed readily in the presence of a variety of catalysts togive polymers of high molecular weights with stannous octoate beingpreferred (refs. 9 to 15).

There are numerous methods for preparing poly(amino acids) (refs. 16, 17and 18) or pseudopoly (amino acids) (refs. 6 and 19).

The noted biodegradable properties of poly-α-hydroxy acids (inparticular those of 50:50 D,L-lactide and glycolide) and poly(aminoacids) has resulted in increased efforts to develop methods forincorporating amino acids into the backbone of α-hydroxy acid polymers(refs. 20 to 25).

Advances have been made in producing copolyesteramides containingα-hydroxy acid sub-units, such as lactide or glycolide, and α-amino acidsub-units, such as glycine or lysine (refs. 22, 23 and 26).

The degradation rate of the biodegradable polymer and the release ratesof encapsulated materials from homopolymers of glycolide, lactide orfrom copolymers of these materials has been shown to be stronglyinfluenced by their molecular weight and structure, such as degree ofcrystallinity and relative hydrophobicity or hydrophilicity.Specifically, microspheres formulated from higher molecular weightpolymers derived from α-hydroxy acids degrade over longer periods oftime than lower molecular weight analogs. Similarly, highly crystallinematerials erode at rates much slower than amorphous analogs. This isrelated to the accessibility of water to the hydrolytically unstableester linkages (ref. 27).

It has been established that random amorphous copolymers composed of 50%D,L-lactide and 50% glycolide exhibit the most advanced degradationrates (refs. 2 and 3) with 50% by weight remaining after approximately 6weeks, when immersed in PBS buffer (pH=7.4).

The copolyesteramides described above are semi-crystalline materialswhich may suffer from prolonged retention at the site of administrationlong after the encapsulated materials are fully released.

Since it would be advantageous to have a polymer that has degraded at ornear the point when the encapsulated material has been fully released,we developed methods for randomly incorporating equal amounts ofD,L-lactide and glycolide into a terpolymer which also containedpseudo-α-amino acid sub-units. A terpolymer of relatively moderatemolecular weight was used to ensure the amorphous terpolymer wouldretain sufficient mechanical strength for processing into films andmicroparticles yet exhibit satisfactory polymer degradation and releaserates for entrapped materials.

The N-protected-L-serine lactone contains an ester bond which may bepolymerized via transesterification catalysts (ref. 6). Additionally ithas been shown that six-membered ring lactones, such as lactide andglycolide, can be copolymerized with four-membered ring propiolactonesby use of insertion/coordination type catalysts/initiators (ref. 13). Itwas expected that efficient transesterification catalysts, such as thosederived from Sn reagents, would be required if relatively sufficientreactivity of all monomer units was to be achieved.

We used the copolymerization of glycolide, D,L-lactide and N-Z-L-serinelactone mediated by stannous octoate. Deprotection of the CBZ group ofthe copolymer or terpolymer can be achieved by various methods. Solidphase catalytic reduction or acid catalysis (ref. 27) are twopossibilities (FIG. 1).

The resultant copolymer or terpolymer can be further elaborated withtargeting moieties such as cell adhesion epitopes, poly ethylene glycol(PEG) ligands for circulation, macrophage stimulators and poly aminoacid grafts as depicted in FIG. 2. A spacer unit may be incorporated,for example, an α-hydroxy acid or a pseudo-α-amino acid unit, and may bereadily derivatised with the appropriate targeting units. The polymer soformed has a molecular weight of from about 5,000 to about 40,000daltons.

Microparticle Formation

The term “microparticle” as used herein refers to any particulatecarrier greater than 1 micron in size which is used for the delivery ofbiologically active materials. The term “microsphere” as used hereinrefers to a microparticle containing one or more active ingredients(e.g. antigens, adjuvants, plasmid DNA).

A flow diagram illustrating the process of microparticle formation asdescribed herein is shown in FIG. 3. In general, the copolymer (PLG,PLGpZS or PLGpS) is solubilized solely or with additional excipientspresent in a compatible solvent, such as dichloromethane, ethyl acetate,acetone or mixtures thereof. Excipients included in the formulation,such as sucrose, mannose, trehalose or gelatin, serve as cryoprotectantsor lyoprotectants. Other materials possessing known adjuvancy, such asBAY R1-005 (BAY) (ref. 29) or tripalmitoyl cysteine (TPC) (ref. 30) or3b[N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) (ref.31) may be included during formulation.

A 1% to 2% copolymer solution of total volume 12 mL is preferablyprepared. To this solution is added 800 μL of phosphate buffered saline(PBS) or 800 μL of antigen solution (concentration typically from 1 to 2mg/mL) in PBS or other stabilizing buffers which may contain additionalexcipients. Other material possessing known adjuvancy, such aspoly[di(carboxylatophenoxy)-phosphazene] sodium salt, (Virus ResearchInstitute, Cambridge Mass.) (PCPP) or cholera toxin or subunits thereofmay be included during formulation. This mixture is then homogenized toform a water in oil emulsion. Once formed, this mixture is dispersedinto 100 mL of a 0.5% to 10.0% aqueous solution containing non-ionicemulsion stabilizers, such as poly vinyl alcohol (PVA), methyl celluloseor Triton X-100. This mixture is immediately homogenized to form a waterin oil in water double emulsion. The average size and polydispersity ofthe resultant droplets can be conveniently measured through use of theCoulter LS-100 light scattering detector. Typical size distributionswhen PBS or Antigen/PBS mixtures are encapsulated range from 1 to 20microns (with the majority less than 10 microns in size). The solvent isthen slowly removed via evaporation with gentle warming to harden theincipient microspheres. Once the solvent is removed the mixture iscentrifuged to collect the microspheres and repeatedly washed withdeionized water to ensure complete removal of residual emulsionstabilizers. The microspheres are then frozen in a dry ice/acetone bathand lyophilized overnight to yield a white freely flowing powder ofmicrospheres (typically 1.0 to 12 microns in size as determined by lightscattering measurements and directly verified via scanning electronmicrography).

The particle generally comprise a polymeric matrix having a particlesize of about 1 to about 50 μM which comprises a polymer having thebiologically-active material entrapped therein, with or without anadjuvant co-entrapped therein. The quantity of biologically-activematerial and adjuvant which may be incorporated into the polymericmatrix may vary widely and may comprise up to about 50 wt % of the totalparticle mass.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of medicine and in particular vaccination, diagnosis andtreatment of infections with pathogens including bacteria and viruses. Afurther non-limiting discussion of such uses is presented below.

Vaccine Preparation

In an embodiment, immunogenic compositions, suitable to be used as, forexample, vaccines, may be prepared from microparticles as disclosedherein. The immunogenic composition containing a biologically activeimmunogenic material can elicit an immune response by the host to whichit has been administered including the production of antibodies by thehost.

The immunogenic composition may be prepared as injectables, as liquidsolutions or emulsions. The microparticles may be mixed withphysiologically acceptable excipients which are compatible with themicroparticles. These may include, water, saline, dextrose, glycerol,ethanol and combinations thereof. The vaccine may further containadditional substances such as wetting or emulsifying agents, pHbuffering agents, or adjuvants to further enhance the effectiveness ofthe vaccines. Vaccines may be administered parenterally, by injectionsubcutaneously or intramuscularly.

Alternatively, the immunogenic compositions comprising microparticlesformed according to the present invention may be delivered in a mannerto elicit an immune response at mucosal surfaces. Thus, the immunogeniccomposition may be administered to mucosal surfaces by nasal, oral(intragastric), buccal or rectal routes. Oral formulations may includenormally employed incipients, such as pharmaceutical grades ofsaccharin, cellulose and magnesium carbonate.

These compositions may take the form of solutions, suspensions, tablets,pills, capsules, sustained release formulations or powders. In order toprotect the microparticles and the encapsulated material containedwithin the core of the microparticle or which is physically mixed withthe microparticles, from gastric acidity when administered by the oralroute, an acidic neutralizing preparation (such as a sodium bicarbonatepreparation) is advantageously administered before, concomitant with ordirectly after administration of the microparticles.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as to be therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of thesubject's immune system to synthesize antibodies, and if needed, toproduce a cell-mediated immune response. Precise amounts ofmicroparticle and material having biological activity required to beadministered depend on the judgement of the practitioner. However,suitable dosage ranges are readily determinable by one skilled in theart and may be of the order of micrograms to milligrams. Suitableregimes for initial administration and booster doses are also variable,but may include an initial administration followed by subsequentadministrations. The dosage of the vaccine may also depend on the routeof administration and thus vary from one host to another.

The polymers of the present invention as applied to vaccineformulations, are also useful for new vaccine strategies, such as withthe use of DNA or antisense RNA. As microparticle carriers, the polymersof the present invention can be prepared to contain DNA coding for agene or genes for an antigenic portion of a virus such as the coreprotein or the envelope protein. As the DNA is released from thecarrier, the host cells take up the foreign DNA, express the gene ofinterest and make the corresponding viral protein inside the cell.Advantageously, the viral protein enters the cells majorhistocompatibility complex pathway which evokes cell-mediated immunity.Standard vaccine antigens in comparison, are taken up into cells andprocessed through the MHC class II system which stimulates antibodyresponses.

The DNA may be in the form of naked DNA which is free of all associatedproteins and which does not require a complex vector system. The desiredgene may be inserted into a vector, such as a plasmid, encapsulatedwithin the microparticle herein described, and injected into the tissuesof a mammal which expresses the gene product and evokes an immuneresponse.

Antisense oligonucleotides can also be used in conjunction with thepolymers of the present invention. Nucleic acid sequences may bedesigned to bind to a specific corresponding mRNA sequence for a knownprotein to inhibit the production of the protein. The microparticlesdescribed herein, can be used to deliver such antisense nucleotides(oligonucleotides or expressed nucleotides) as a therapeutic strategyfor the treatment of various immunological diseases as well ashypertension, cardiovascular disease and cancer.

The slow-release characteristic of the polymer microparticles developedherein also has use in the field of pharmacology where themicroparticles can be used to deliver pharmacological agents in a slowand continual manner. A wide range of drugs, such as anti-hypertensives,analgesics, steroids and antibiotics, can be used in accordance with thepresent invention to provide a slow release drug delivery system.

The polymer in the form of a film having a biological agent entrapped orphysically admixed thereto, may also have use as a coating for surgicalimplants and devices. For example, the polymer as a film havingantibiotic incorporated therein can be used to coat surgically implantedcatheters in order to provide continual slow-release of antibiotics tocombat infection.

The microparticle carrier may also be useful as a diagnostic agent.Together with the appropriate antibody, imaging agents can beincorporated with the microparticles. In this manner diseased tissuescan be targeted and imaged in order to identify or monitor the clinicalcourse of a disease.

The polymers, as microparticles, also have use in diagnostic kits whenused in conjunction with appropriate antibodies.

EXAMPLES

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Changes in the form and substitution of equivalents are contemplated ascircumstances may suggest or render expedient. Although specific termshave been employed herein, such terms are intended in a descriptivesense and not for purposes of limitations.

Methods of chemistry, organic chemistry, polymer chemistry, proteinbiochemistry and immunology used but not explicitly described in thisdisclosure and these Examples are amply reported in the scientificliterature and are well within the ability of those skilled in the art.

Example 1

This Example illustrates the preparation of N-Z-L-Serine β-Lactone.

The preparation of this cyclic N-protected amino acid lactone was basedon a modified procedure in which an N-protected-α-amino acid is reactedto yield cyclized pseudo-α-amino acid monomer (ref. 6). All glasswarewas pre-dried overnight in an oven set at 120° C. Prior to use it wascooled in a vacuum desiccator and purged under a stream of dry nitrogenfor 10 minutes.

To a 1 L three necked round bottomed flask under nitrogen was addedtriphenylphosphine (TPP; Aldrich;

7.87 mL; 50 mmol; FW: 174.16). To this was added 200 mL of anhydrousacetonitrile (CH₃CN; Aldrich): anhydrous tetrahydrofuran (THF; Aldrich)solution (volume ratio 85:15) via syringe and stirred until the solidTPP was dissolved. To this solution diethyl azodicarboxylate (DEAD;Aldrich; 7.87 mL; 50 mmol; FW: 262.29) was added via syringe and thesolution stirred at room temperature for 30 minutes. The solution wasthen cooled to about −45° C. to −48° C. by immersing the reaction vesselin an acetonitrile/dry ice bath. Once the internal temperature of thesolution reached about −45° C., a solution of N-Carbobenzyloxy-L-Serine(N-CBZ-L-Serine; Sigma; 11.94 g; 49.8 mmol; FW: 239.2) in 200 mL ofanhydrous CH₃CN:THF (volume ratio 85:15) was slowly added via droppingfunnel over a period of 1 hour. The temperature of the solution wasmaintained at about −45° C. during the addition and allowed to slowlywarm to room temperature once the addition was complete with continuousstirring overnight. This reaction results in the formation of an esterbond between the serine hydroxyl side chain and the carboxylic acid inthe presence of the CBZ protected α-amino group. Upon completion of thereaction the solvents were removed via evaporation (35° C. to 45° C.).This yields a yellow oil/slurry (˜35 g). To this slurry was added 50 mLof dichloromethane:ethyl acetate (volume ratio 85:15) solution whichresults in the precipitation of 1,2-dicarbethoxyhydrazine byproduct.This material was removed by filtration under vacuum followed by solventremoval via evaporation. The above procedure can be repeated to furtherremove residual byproducts. The waxy solid crude material was thenpurified via silica gel column chromatography with eluent 85:15dichloromethane:ethyl acetate as solvent. The product serine lactone canbe identified via thin layer chromatography as this material has anR_(f) of 0.75 on silica plates eluted with 85:15 dichloromethane:ethylacetate when stained with a 1 M H₂SO₄ solution and is also UV visible.The product was recrystallized from ethyl acetate:hexane (˜1L), filteredand dried in vacuo.

A clean white solid is obtained in 40% yield after recrystallizationwith a melting point (Tm=133-134° C.) and all other physical parameters(NMR, IR, mass spectroscopy, elemental analysis) conforming to thatpreviously demonstrated (ref. 6).

Example 2

This Example illustrates the preparation of the copolymerpoly-D,L-Lactide-co-Glycolide-co-pseudo-Z-Serine Ester (PLGpZS) as shownin FIG. 1.

Glassware was pre-dried overnight. Prior to use it was cooled in avacuum desiccator. Additionally the polymerization vessel (glass ampule)must be siliconized (SurfaSil; Pierce; 2% solution in toluene) and alltransfer reactions and additions of reagents and monomers topolymerization vessel must be conducted in a glove box maintained undera dry nitrogen environment.

Prior to polymerization the D,L-lactide(2,6-dimethyl-1,4-dioxane-2,5-dione; Aldrich; FW: 144.13) and glycolide(Boehringer Ingelheim; FW: 116.096) was recrystallized from anhydrousethyl acetate in the glove box and dried in vacuo for about 2 days. Oncefully dried the monomers can be stored in the glove box with the freshlyrecrystallized serine lactone (stored at 0° C.) of Example 1 broughtdirectly into the glove box. All monomers and catalyst/initiators wereweighed and transferred to glass ampules within the glove box.

The total combined mass of monomer transferred to the ampoule typicallyranges from 1 g to 5 g with the molar ratio ofD,L-lactide:glycolide:serine lactone ranging from 42.5:42.5:15.0 to49.0:49.0:2.0. A molar ratio of D,L-lactide:glycolide:serine lactone of47.5:47.5:5.0 was used in the preferred embodiment. A stock solution ofcatalyst (stannous 2-ethylhexanoate (Sn(Oct)₂; Sigma; FW 405.1, 1.25g/mL) in anhydrous chloroform (Aldrich) was prepared in the glove boxand added via microsyringe to the glass ampule (molar ratio of catalystto monomer=1/1000). A stir bar was also placed in the ampule. A greasedground glass joint valve was placed on the ampule to preserve the inertenvironment during removal from the glove box. The ampule was thendirectly placed on a vacuum line with slow removal of chloroform byevaporation. The ampules were then placed in an oil bath at ca. 120° C.to bring all reagents into the melt followed by flame sealing andplacement in a thermoregulated oven at 120° C. for 28 hours. Afterreaction the ampules are quenched by placing in liquid nitrogen andstored at −20° C. until further work up. The ampule was cracked andcrude polymer recovered by dissolving in chloroform. The solvent wasremoved by evaporation and crude polymer dried in vacuo to give an ambercrystalline material (yield 80% to 90%).

The polymer was purified by dissolving in chloroform, filtering offinsoluble material and precipitating into hexane (Aldrich). The polymerwas recovered by filtration and this procedure was repeated to ensurethe complete removal of unreacted monomer. The polymer was dried invacuo for about 2 days to give a clean white powder in 30% to 35%overall yield after second precipitation. The molecular weight of thismaterial was dependent on reaction time with typical values ofMw=17,000-22,000, Mn=6,500-8,000. Differential scanning calorimetry(DSC) analysis indicates a single transition indicative of a randomamorphous polymer. Glass transitions (Tg) range from 39° C. to 43° C.dependent on the molecular weight of the material obtained. The serinecontent of the polymer was determined by amino acid analysis (AAA)diagnostic for the phenylthio isocyanate serine derivative obtained byhydrolysis of the polymer, ¹H NMR (integration of aromatic residues ofthe CBZ protecting group on serine relative to the glycolide andD,L-lactide sub-units) and elemental analysis (nitrogen present only inthe side chain of serine). The AAA analysis typically indicated 1.7% to2.1% serine content with the ¹H NMR analysis indicating 2.0% to 2.5% andthe elemental analysis indicating 2.4% to 3.4% serine respectively. IRanalysis of the polymer was diagnostic for the presence of ester,carbamate and hydroxyl groups.

¹H NMR also allowed for the determination of the relative incorporationefficiencies of all monomer components under the stated reactionconditions. Typical ratios of D,L-lactide:glycolide:Z-serine found inpurified polymer are reproducibly 52.0% to 54.0% D,L-lactide, 41.0% to43.5% glycolide and 2.0% to 2.5% Z-serine respectively.

¹H NMR and ¹³C NMR signal intensities for resonances unique to glycolideor D,L-lactide are well resolved from each other and sensitive tosequence effects. From the observed patterns a random sequencedistribution is supported.

Example 3

This Example illustrates the preparation of the copolymerpoly-D,L-Lactide-co-Glycolide-co-pseudo-Serine Ester (PLGpS) as shown inFIG. 1.

All glassware was pre-dried overnight. Prior to use it was cooled in avacuum desiccator and purged under a stream of dry nitrogen for 10minutes. All reactions were conducted under inert atmosphere of drynitrogen.

To a 2 necked 100 mL round bottomed flask equipped with a stir bar wasplaced 400 mg of polymer (PLGpZS) To this was added a 10 mL solution of30% hydrogen bromide in acetic acid (Aldrich; FW: 80.92) which wassufficient for slurry formation. The slurry was stirred for 30 to 45minutes and quenched by dropwise addition of anhydrous diethyl ether(Aldrich) followed by anhydrous methanol (Aldrich). This results inpolymer precipitation which was then isolated by vacuum filtration. Thecrude polymer precipitate was washed with diethyl ether andreprecipitated from chloroform:hexane. The purified polymer was dried invacuo for about 2 days to give a clean white powder in 50% to 60%overall yield. The molecular weight ranged from Mw=15,000-18,000,Mn=5,000-6,500. The rate of deprotection of the CBZ group is faster thanthe competitive cleavage of the ester backbone with HBr/Acetic acid.However, under these conditions there is broadening of the molecularweight distribution and reduction in the molecular weight of the productas a consequence of using this reagent. This trend can be reduced byconducting the reaction for shorter time intervals or eliminated byremoving the protecting group via hydrogenation using hydrogen in thepresence of palladium on charcoal. DSC analysis indicates a singletransition indicative of a random amorphous polymer. Glass transitions(Tg) range from 42° C. to 45° C. depending on the molecular weight ofthe material obtained. The serine content of the polymer was determinedby amino acid analysis (AAA) diagnostic for the phenylthio isocyanateserine derivative obtained by hydrolysis of the polymer and elementalanalysis (nitrogen present only in the side chain of serine) The AAAanalysis typically indicated 1.4% to 1.7% serine content and theelemental analysis indicating 2.0% to 2.7% serine respectively. IRanalysis of the polymer was diagnostic for the presence of ester, amineand hydroxyl groups.

¹H NMR for residual protected polymer indicated that greater than 90% ofthe N-carbobenzyloxy groups were successfully removed. With shorterreaction times the extent of deprotection is concomitantly reduced.Typical ratios of D,L-lactide:glycolide:Z-serine:serine found inpurified polymer are reproducibly 53.0% to 55.0% D,L-lactide, 40.0% to43.0% glycolide, 0.15% to 0.25% Z-serine and 1.7% to 2.1% serinerespectively.

Example 4

This Example illustrates the production of a film from the copolymerssynthesized in Examples 2 and 3.

To produce the film, 50 mg of poly-D,L-Lactide-co-Glycolide (PLG)(Mw=31,000), poly-D,L-Lactide-co-Glycolide-co-pseudo-Z-Serine Ester(PLGpZS) (Mw=20,000) or poly-D, L-Lactide-co-Glycolide-co-pseudo-SerineEster (PLGpS) (Mw=19,000) was weighed out and placed in a 10 mL beaker.Anhydrous chloroform (1 mL) was added to dissolve the copolymer. Thissolution was filtered and added dropwise to a microscope slide placed ina petri dish. The petri dish was then covered with a 250 mL beaker toensure slow evaporation over 48 hours. The resultant films weretranslucent and contact angle measurements performed using a goniometergave average values of 75° for PLG, 75° for PLGpZS and 68.2° for PLGpSrespectively. Thus the PLG and PLGpZS copolymers are of comparablehydrophobicity with the PLGpS copolymer proving to be slightly morehydrophilic and of higher surface energy.

Example 5

This Example illustrates the process of microparticle formationencapsulating PBS or antigen/PBS (microsphere formation).

A flow diagram illustrating the process of microparticle formation asdescribed herein is shown in FIG. 3.

Specifically, 100 mg of copolymer was added to 12 mL of dichloromethane.To this was added 800 μL of phosphate buffered saline (PBS) solution or800 μL of non-proteolytic Hin-47 analog (concentration typically from 1to 2 mg/mL) in PBS. This mixture was then homogenized (20 seconds at6,000 rpm). Once formed this mixture was dispersed into 100 mL of a 1.0%aqueous solution of poly vinyl alcohol (PVA) and immediately homogenized(40 seconds at 8,000 rpm) to form a water in oil in water doubleemulsion. Typical size distributions when PBS or a typical antigen(Hin-47/PBS) is used as encapsulant are depicted in FIG. 4. Polydispersemicroparticles (with the majority less than 10 microns in size) wereformed under these conditions. The solvent was then slowly removed viaevaporation and the microspheres collected by centrifugation. Theparticles were washed (5×) with deionized water and then frozen in a dryice/acetone bath and lyophilized overnight to yield a white freelyflowing powder of microspheres (typically 1.5 to 10 microns in size asdetermined by light scattering measurements and directly verified viascanning electron micrography). A representative scanning electronmicrograph for PLGpZS or PLGpS microspheres encapsulating PBS is shownin FIG. 5. A representative scanning electron micrograph for PLGpZS orPLGpS microspheres encapsulating a typical antigen (non-proteolyticHin-47 analog) in PBS is shown in FIG. 6.

By the method stated above microparticles containing several differentantigen(s) and/or antigen(s)+adjuvant have been prepared (see Tables 1and 5).

Example 6

This Example illustrates the microparticle core loading efficiency andantigen epitope recovery from such microparticles.

Two variations of the same method were employed to determine the antigencontent or “core loading” of the microparticles isolated. Amino acidanalysis was performed on the hydrosylates of microparticles obtained byeither acid hydrolysis (6M HCl) of the solid particles or by base/SDShydrolysis (0.1N NaOH/1% SDS) followed by neutralization with 0.1N HCl.Alternatively, the solid microspheres can be dissolved in DMSO acompatible solvent solubilizing both polymer and protein and amino acidanalysis performed directly on the lyophilized sample. The acid or basemediated hydrolysis proved to be the preferred method giving the mostreproducible results (+/−5%). Where available a validated Enzyme LinkedImmunosorbant Assay (ELISA) polyclonal assay was performed on thehydrosylates to determine the epitope equivalence.

Specifically, for the quantitation of non-proteolytic Hin-47 analogantigen by ELISA the non-proteolytic Hin-47 analog antigen was capturedon affinity purified guinea pig anti-Hin47 coated microtitre wells (Add50 μL of a 2 μg/mL solution of non-proteolytic Hin-47 analog antigen perwell), which have been blocked with 5% skim milk in PBS. The antigenpresent was detected by an affinity purified rabbit anti-Hin-47 followedby horse radish peroxidase F(abs)2, donkey anti-rabbit IgG. To developthe color of this reaction 100 μL of the substrate H₂O₂ (9 parts) in thepresence of tetramethylbenzidine (TMB) (1 part), and the reactionprogress terminated by addition of 50 μL of a 2M sulphuric acid solutionto each well. The intensity of the color (read at 450 nm) was directlyproportional to the amount of non-proteolytic Hin-47 analog in the well.The concentration of non-proteolytic Hin-47 analog in each test samplewas derived by extrapolation from a standard curve which was obtained bymultiple dilutions of a reference sample. All samples were analyzed induplicate. The amino acid analysis for core loading and Hin-47 specificpolyclonal ELISA analysis of epitope recovery for microparticlesencapsulating non-proteolytic Hin-47 analog or non-proteolytic Hin-47analog plus BAY R1-005 within PLG, PLGpZS and PLGpS copolymers is shownin Table 2. The core loadings for microparticles prepared solely fromHin-47 range from 20% to 43% with from 10% to 31% of the epitopepreserved during formulation. The core loadings for microparticlesprepared from non-proteolytic Hin-47 analog in the presence of BAYR1-005 range from 26% to 59% (an absolute increase of 6% to 16%) withfrom 20% to 39% (an absolute increase of 4% to 18%) of the epitopepreserved during formulation. Thus formulation in the presence of BAYR1-005 concomitantly increases the loading efficiency and serves toprotect the epitope.

The amino acid analysis of core loading for microparticles encapsulatingrD-15 within PLG, PLGpZS and PLGpS copolymers is shown in Table 3. Coreloadings ranging from 39% to 44% were obtained. This protein wasmembrane derived and thus more hydrophobic than the non-proteolyticHin-47 analog of the previous example. Increased encapsulationefficiencies relative to non-proteolytic Hin-47 analog of Example 10were routinely observed when using this antigen.

The amino acid analysis for core loading and epitope recovery formicroparticles encapsulating a cocktail of non-proteolytic Hin-47 analogand rD-15 (1:1 cocktail) within PLG, PLGpZS and PLGpS copolymers isshown in Table 3. It was expected that coencapsulation ofnon-proteolytic Hin-47 analog in the presence of rD-15 would result inhigher overall loading efficiencies. Core loadings ranging from 35% to62% were obtained with from 8% to 26% of the non-proteolytic Hin-47analog epitope preserved during formulation. The overall loadingefficiency of the more hydrophilic component (non-proteolytic Hin-47analog) was increased by coencapsulation with rD-15 when formulated withPLGpZS or PLGpS copolymers. This effect was not observed when PLG wasused. Irrespective of copolymer formulation similar non-proteolyticHin-47 analog epitope recovery as compared with Example 10 was obtained.

The amino acid analysis of core loading for microparticles encapsulatingFlu X-31 or Flu X-31 and BAY R1-005 within PLG, PLGpZS and PLGpScopolymers is shown in Table 4. Core loadings ranging from 26% to 40%were obtained with Flu X-31 and for Flu X-31 in the resence of BAYR1-005 core loadings ranging from 31% to 53% were obtained. Thus, anabsolute increase of 5% to 13% was obtained when formulating in thepresence of BAY R1-005. The only exception was the formulation of FluX-31 with BAY R1-005 and PLGpS where an absolute decrease of 9% wasobserved.

A core loading of 21% for PLGpS microparticles encapsulating Flu A-Texaswas obtained. For PLGpS microparticles encapsulating Flu A-Texas in thepresence of BAY R1-005 a core loading of 32% was obtained.

When a different strain of Flu was employed (A-Texas) with BAY R1-005and PLGpS copolymer the tendency to increase the encapsulationefficiency was once again observed. When formulating PLGpS with FluA-Texas in the presence of BAY R1-005 an absolute increase of 11% wasobtained. This suggests that the formulation for PLGpS and proteins inthe presence of BAY R1-005 must be optimized in each case. As in Example10, the addition of the coadjuvant BAY R1-005 has resulted in higherloading efficiencies.

The amino acid analysis of core loading for microparticles encapsulatingFlu (trivalent) vaccine or Flu (trivalent) vaccine and BAY R1-005,DC-Chol or PCPP within PLGpS copolymers is shown in Table 6. Excellentcore loading efficiencies ranging from 72% to 104% were obtained usingthe procedure of Example 5. The improvement in encapsulationefficiencies is a general result observed when the optimum concentrationof protein is used to form the primary emulsion. In this case, theoptimum concentration was experimentally determined to be about 0.5mg/mL.

The amino acid analysis for core loading and rUrease specific polyclonalELISA analysis of epitope recovery for microparticles encapsulatingrUrease or rUrease plus DC-Chol, PCPP or CT-X within copolymers is shownin Table 7. Epitope recovery is defined as the difference between thetotal protein obtained by amino acid analysis and total protein obtainedby polyclonal ELISA expressed as a percentage. The core loadings formicroparticles prepared solely from rUrease is about 46% with about 72%of the epitope recovered during formulation. Similarly the core loadingsfor microparticles prepared from rUrease in the presence of DC-Chol isabout 44% with about 69% epitope recovered. Slightly higher totalprotein was determined for microparticles prepared from rUrease in thepresence of PCPP. A core loading of 67% and epitope recovery of about55% was found for this combination. For the rUrease/CT-X combination,the total core loading for rUrease could not be estimated by amino acidanalysis since both rUrease and CT-X contribute to this value. Apolyclonal ELISA assay for CT-X was developed and microparticlehydrosylates assayed for total rUrease and CT-X separately. An epitoperecovery of 53% was found for rUrease and 59% for CT-X respectively.SDS-PAGE and Western blots performed on these microparticle hydrosylatesconfirmed the presence of non-degraded rUrease and CT-X.

Suitable controls were performed to ensure that the polyclonal ELISAassay for rUrease was unaffected by additives, such as DC-Chol and PCPP.In the case of PCPP, the assay was often problematic and variable. Nosimilar problem was found when assaying rUrease in the presence ofDC-Chol.

Example 7

This Example illustrates the in vitro release rates and total cumulative% recovery of protein for a model antigen (non-proteolytic Hin-47analog) encapsulated within PLG (used as a control for comparisonpurposes) and synthesized copolymers PLGpZS and PLGpS.

The PLG formulated into microspheres containing antigen was determinedto be of molecular weight; Mw=26,000 with a 50:50 ratio ofD,L-Lactide:Glycolide. This material is of similar constitution andmolecular weight to that obtained for copolymers PLGpZS and PLGpS ofExamples 2 and 3. In a typical experiment, 14 mg of microspheres (coreloading of non-proteolytic Hin-47 analog for PLG=2.8 μg/mg, PLGpZS=5.7μg/mg and PLGpS=2.5 μg/mg as determined by amino acid analysis of themicrosphere hydrosylates) was placed in a 2 mL eppendorf tube to whichwas added 1.0 mL of PBS (pH=7.4). The tube was then placed in athermoregulated oven maintained at 37° C. without agitation. At varioustime intervals the PBS solution was extracted and analyzed for totalprotein via amino acid analysis. After each extraction the PBS solutionwas replaced with continued sampling to a maximum of 90 days. In controlexperiments, the majority of the microspheres (>80% by weight) preparedfrom PLG or PLGpS copolymers were consumed by day 45. The degradation ofmicrospheres formulated from PLGpZS copolymers was observed to besomewhat slower requiring 60 days to erode to essentially the sameextent.

Similar antigen release trends were observed with each group ofmicroparticles wherein small amounts of protein were released over thefirst few days. This diminished to near undetectable limits up to day 14whereafter the protein release rate steadily increased to a maximum atabout day 30. Subsequent to this time, the release of protein fell tolevels again approaching the limit of detection. The total % cumulativerecovery of protein from these samples ranged from 40% to 65% relativeto the respective core loadings of each group of microspheres.

FIG. 7A illustrates the release rate at specific time points for eachsample and FIG. 7B shows the % cumulative release for each sampleexamined. It is evident that under these conditions the best recovery ofprotein from microspheres follows the order PLGpS>PLG>PLGpZS althoughthe core loading for the PLGpZS microparticles was approximately twicethat of the PLG or PLGpS analogs and this may influence the releaserate. In addition, this matrix has been shown to degrade at a slowerrate and thus there may be residual material within the microparticles.Evidence for this can be seen in FIG. 7B whereby a marginal increase inprotein recovery was observed from day 60 to day 90 for PLGpZSmicroparticles. Over this same time interval, protein recovered from PLGor PLGpS microparticles was essentially non-existent.

In control experiments, supernatant solutions of PBS obtained fromperiodic extractions of PBS loaded PLG, PLGpZS or PLGpS microparticlesincubated at 37° C. were monitored for pH changes. It is known that theerosion process for PLGA matrices is accompanied by changes in the pHmicroenvironment within the microparticle (ref. 36). This can have aserious effect on protein stability as pH induced conformational changescan ensue as a consequence of these changes. An indication of themagnitude of these changes can be obtained by monitoring the pH of thesurrounding medium. We have found that incorporation of smallpercentages of amino acid sub-units within the copolymer can retard thisprocess. This may be of importance during the release phase for proteinssensitive to acidic pH. Specifically when incubating ˜10 mg ofmicroparticles in 1.2 mL of PBS buffer (pH=7.4) it requiredapproximately 16 days for supernatant extractions of PLG (Mw=26,000daltons) to fall below 5 pH 5.0. By analogy the pH of supernatantextractions obtained from PLGpZS (Mw=20,000 daltons, approximately 2.0%serine incorporated), or PLGpS (Mw=18,000 daltons, approximately 1.7%serine incorporated) was determined to be approximately 5.5 to 6.2respectively. The degradation rates for PLG and PLGpS examined in thisstudy were similar (as measured by mass loss of matrix) whereas thePLGpS matrix degraded at a reduced rate.

Thus the in vitro release study demonstrates that a single dose delayedrelease delivery system can be achieved through use of polymericmicroparticles formulated from PLGpZS or PLGpS copolymers. In additionas pH changes with matrix erosion can have a deleterious effect on theprotein stability it may be advantageous to employ matrices derived frompseudo-serine ester such as PLGpZS or PLGpS wherein there exists somebuffering capacity for these changes during the protein release phase.

Example 8

This Example illustrates the immunogenicity of non-proteolytic Hin-47analog encapsulated or physically mixed with microparticles in micewhich were immunized subcutaneously.

To examine the immunogenicity of non-proteolytic Hin-47 analog entrappedin PLG, PLGpZS and PLGpS microparticles formed in accordance with thepresent invention, groups of five, 6 to 8 week old female BALB/c mice(Charles River Breeding Laboratories, Wilmington, Mass.) were immunizedsubcutaneously (S.C.) with the following amounts of antigen in 250 μL ofPBS (pH 7.4) on days 1 and 35: PLG, PLGpZS and PLGpS microparticlesprepared as described in Example 5 containing 0.2 μg or 0.6 μg ofnon-proteolytic Hin-47 analog (FIG. 8A); and PLG microparticles preparedas described in Example 5 physically mixed with 0.8 μg or 2.5 μg ofnon-proteolytic Hin-47 analog (FIG. 8B).

The mice showed no gross pathologies or behavioral changes afterreceiving microparticles that contained encapsulated non-proteolyticHin-47 analog or microparticles that were physically mixed withnon-proteolytic Hin-47 analog. Sera were obtained on days +10, +24, +35,+45 and +60 and were evaluated for the presence of anti-Hin-47 IgGantibodies by antigen specific ELISA. All samples were analyzed induplicate. Microtiter plate wells were incubated overnight at roomtemperature with 100 μL of 0.2 μg/mL non-proteolytic Hin-47 analog in0.05M carbonate-bicarbonate buffer (pH 9.0). The plates were washed withPBS+0.05% Tween 20 (operationally defined as washing buffer). Wells wereincubated with 200 μL of 5% skim milk (operationally defined as blockingbuffer). After washing with PBS+0.05% Tween 20, the plates wereincubated for 1 h at 37° C. with 100 μL of sample serially diluted inblocking buffer. Wells were washed with PBS+0.05% Tween 20 and 100 μL ofHRP-conjugated antibody (goat anti-mouse IgG (H+L) (Jackson), sheepanti-mouse IgG1 (Serotec), goat anti-mouse IgG2a (Caltag) or goatanti-mouse IgG2b (Caltag) in blocking buffer was added to each well.After 1 hour incubation at 37° C., the wells were washed five times withPBS+0.05% Tween 20 and 100 μL of freshly prepared colorizing substrate[H₂O₂ (9 parts) and TMB (1 part)] is added to each well. After 5 minutesincubation in the dark at room temperature the reaction is stopped byadding 50 μL of a 2M H₂SO₄ solution and the optical density of the fluidin each well was determined at 450 nm using a microplate reader. Anormal mouse sera pool was used to establish baseline optical densityvalues in the assay. Hyperimmune mouse Hin-47 antiserum was used as apositive control. Pre-immune sera was used as negative control.

For serum IgA analysis the above procedure was conducted with thefollowing modification. Microtiter plate wells were incubated overnightat room temperature with 100 μL of 1.3 pg/mL non-proteolytic Hin-47analog in 0.05M carbonate-bicarbonate buffer (pH=9.0), and 100 μL of HRPconjugated rabbit anti-mouse IgA (ZYMED, CA) at 0.06 μg/mL was added toeach well.

For secretory IgA analysis, the above procedure was conducted with thefollowing modification. 100 μL of HRP conjugated rat anti-mouse IgA(Biotin-Pharmigen) at 0.06 μg/mL was added to each well.

The serum antibody titres following immunization are shown in FIGS. 8Aand 8B. The results of immunizations indicate that antigen presented tothe immune system entrapped in PLG, PLGpZS or PLGpS microparticles (FIG.8A) was substantially more immunogenic than soluble antigen at dosessub-optimal to that required with soluble antigen alone. In addition, nodose dependence was observed with encapsulated non-proteolytic Hin-47analog of dose 0.2 μg or 0.6 μg, whereas a marginal increase in titrewas found with soluble non-proteolytic Hin-47 analog of dose 0.8 μg or2.5 μg respectively.

The results of immunizations indicate that antigen presented to theimmune system when physically mixed with PLG microparticles (FIG. 8B) ismarginally more immunogenic than soluble antigen at similar dose to thatgiven with soluble antigen alone (0.8 μg or 2.5

82 g). However, administration of antigen in soluble form or admixedwith microparticles elicits a response several orders of magnitude lessthan that seen for antigen encapsulated within the microparticlesdemonstrating the advantages of encapsulation over soluble or physicallymixing alone.

Interestingly, the IgG subtype profile (FIG. 8C) for pooled serumobtained from the bleeds on day 35 and 60 for microparticle encapsulatednon-proteolytic Hin-47 analog, microparticles physically mixed withnon-proteolytic Hin-47 analog or soluble non-proteolytic Hin-47 analogshows that by day 35 IgG1 was the dominant subtype with some IgG2bdetected irrespective of formulation. By day 60 it was evident that theIgG subtypes induced by non-proteolytic Hin-47 analog encapsulatedwithin microparticles had undergone class switching such that IgG1,IgG2a and IgG2b are more equally represented. The IgG subtypes inducedby non-proteolytic Hin-47 analog physically mixed with particles or insoluble form by day 60 was nominally the same as that determined for day35 with the IgG1 subtype dominant.

Thus, it can be concluded that the qualitative nature of the immuneresponse mediated by microparticles encapsulating antigen issubstantially different than that obtained by physically mixing withmicroparticles or by soluble antigen alone. The presence of IgG2asubtype in the BALB/c mouse model is generally accepted to be indicativeof a TH₁ pathway, and IgG1 indicative of a TH₂ pathway. It is evidentthat via the subcutaneous route the TH₂ pathway is favored for solubleantigen or for antigen physically mixed with microparticles, whereaswith antigen encapsulated within microparticles a more balanced TH₁/TH₂response is attainable.

Example 9

This Example illustrates the immunogenicity of non-proteolytic Hin-47analog entrapped in PLG, PLGpZS and PLGpS microparticles formed inaccordance with the present invention, in mice immunizedintragastrically.

Groups of five, 6 to 8 week old female BALB/c mice (Charles RiverBreeding Laboratories, Wilmington, Mass.) were immunizedintragastrically (I.G.) with the following amounts of antigen in 250 μLof 0.15 M NaHCO₃ (pH 9.0) on days 1, 7, 14 and 57: PLG, PLGpZS and PLGpSmicroparticles prepared as described in Example 5 containing 4.0 μg ofnon-proteolytic Hin-47 analog; and PLG, PLGpZS and PLGpS microparticlesprepared as described in Example 5 physically mixed with 4.0 μgnon-proteolytic Hin-47 analog (FIG. 9A).

The mice showed no gross pathologies or behavioral changes afterreceiving microparticles that contained encapsulated non-proteolyticHin-47 analog or microparticles that were physically mixed withnon-proteolytic Hin-47 analog. Sera were obtained on days +13, +35, +56and +78 and were evaluated for the presence of anti-Hin-47 IgG and IgAantibodies by antigen specific ELISA as described in Example 8.

Sera and intestinal washes were examined for the presence ofHin-47-specific antibodies. To detect and quantify anti-Hin-47 IgG andsIgA in the intestinal lumen, mice were sacrificed by cervicaldislocation, their small intestines removed and examined for thepresence of antigen-specific antibodies. Individual small intestineswere detached from the pyloric sphincter to the caecum and everted overcapillary tubes. The everted intestines were incubated in 5 mL of icecold enzyme inhibitor solution (0.15 M NaCl, 0.01 M Na₂HPO₄, 0.005 MEDTA, 0.002 M PMSF, 0.05 U/mL Aprotinin, and 0.02% v/v NaN₃) for 4hours. Intestines were removed and the supernatants clarified bycentrifugation (1000 xg, 20 minutes) and stored at 0° C. until assayed.Anti-Hin-47 IgG and sIgA titres in samples were determined byHin-47-specific ELISA as described above but a goat anti-mouse IgAantiserum was used in place of the goat anti-mouse IgG antiserum.

The serum IgG Hin-47-specific antibody titres following I.G.immunization is shown in FIG. 9A. These results indicate that an antigen(non-proteolytic Hin-47 analog) incorporated into PLG, PLGpZS or PLGpSmicroparticles was substantially more immunogenic than soluble antigenof similar dose (4 μg) and better than antigen that was physically mixedwith microparticles when delivered by the intragastric route. It wasexperimentally determined that a dose of soluble antigen (20 μg) whichwas five times that which was administered encapsulated withinmicroparticles (4 μg) was required to elicit an essentially equivalentresponse. This result is atypical for most proteins as there are manyexamples where in excess of 100 μg of antigen is required to elicit anysignificant serum IgG antibody response via the intragastric route.

The IgG subtype profile (FIG. 9B) for pooled serum obtained from thebleeds on day 56 and 78 for microparticle encapsulated non-proteolyticHin-47 analog, microparticles physically mixed with non-proteolyticHin-47 analog or soluble non-proteolytic Hin-47 analog shows a similartrend as that observed in Example 8. The IgG1 subtype was dominant whenantigen was delivered in soluble form or when physically mixed withmicroparticles. Non-proteolytic Hin-47 analog encapsulated withinmicroparticles exhibits a more balanced profile with IgG1 and IgG2a moreequally represented. Thus via the intragastric route with antigenencapsulated within microparticles a more balanced TH₁/TH₂ response wasattainable.

FIG. 9C shows the results for anti-Hin-47 IgA antibody responsesobtained from bleeds on day 78. With soluble antigen at 4 μg per dose nodetectable serum IgA was found, however at 20 μg per dose a fewresponders were observed. A single significant response was observedwith antigen encapsulated within PLG microparticles and moderateresponses observed for antigen encapsulated within PLGpZS or PLGpSmicroparticles. Similarly modest response was seen for antigenphysically mixed with PLG, PLGpZS or PLGpS microparticles. The highestaverage levels of serum IgA were obtained for antigen encapsulatedwithin PLGpS microparticles. The intestinal lavage conducted on day 78revealed minimal levels of IgG or sIgA specific for Hin-47 analog in themucosal secretions obtained from non-proteolytic Hin-47 analogencapsulated within PLG, PLGpZS or PLGpS microparticles. This is likelydue to the very low levels of encapsulated antigen administered in thisexperiment (4 μg per dose) as oral doses of antigen ranging from 30 μgto 100 μg are usually required to elicit a significant mucosal responsein the absence of any other mucosal adjuvants.

Example 10

This Example illustrates the immunogenicity of non-proteolytic Hin-47analog entrapped in PLG, PLGpZS and PLGpS microparticles formed inaccordance with the present invention, in mice immunized intranasally.

Groups of five, 6 to 8 week old female BALB/c mice (Charles RiverBreeding Laboratories, Wilmington, Mass.) were immunized intranasally(I.N.) with the following amounts of antigen in 25 μL of PBS (pH 7.4) ondays 1, 7, 14 and 57: PLG, PLGpZS and PLGpS microparticles prepared asdescribed in Example 5 containing 4.0 μg of non-proteolytic Hin-47analog; and PLG, PLGpZS and PLGpS microparticles prepared as describedin Example 5 physically mixed with 4.0 μg non-proteolytic Hin-47 analog(FIG. 10A).

The mice showed no gross pathologies or behavioral changes afterreceiving microparticles that contained encapsulated non-proteolyticHin-47 analog or microparticles that were physically mixed withnon-proteolytic Hin-47 analog. Sera were obtained on days +13, +35, +56and +78 and were evaluated for the presence of anti-Hin-47 IgG and IgAantibodies by antigen specific ELISA as described in Example 8.

Sera and lung lavage washes were examined for the presence ofHin-47-specific antibodies. To detect and quantify anti-Hin-47 IgG andsIgA in the respiratory tract, mice were sacrificed by cervicaldislocation, a small incision was made in the trachea and a tubeinserted through the trachea into the lungs where 400 μL of an enzymeinhibitor solution (0.15 M NaCl, 0.01 M Na₂HPO₄, 0.005 M EDTA, 0.002 MPMSF, 0.05 U/mL Aprotinin, and 0.02% v/v NaN₃) was injected. Thissolution was then withdrawn, placed on ice and the supernatantsclarified by centrifugation (1000 xg, 20 minutes) and stored at 0° C.until assayed. Anti-Hin-47 IgG and sIgA titres in samples weredetermined by Hin-47-specific ELISA as described in Example 8.

The serum IgG Hin-47-specific antibody titres following I.N.immunization is shown in FIG. 10A. These results indicate that anantigen (non-proteolytic Hin-47 analog) incorporated into or physicallymixed with PLG, PLGpZS or PLGpS microparticles was more immunogenic thansoluble antigen of similar dose (4 μg). Essentially equivalent responsewas obtained for antigen that was encapsulated or physically mixed withmicroparticles by the intranasal route.

The IgG subtype profile (FIG. 10B) for pooled serum obtained from thebleeds on day 56 and 78 for microparticle encapsulated non-proteolyticHin-47 analog, microparticles physically mixed with non-proteolyticHin-47 analog or soluble non-proteolytic Hin-47 analog illustrated atrend different from that shown in Example 8 or Example 9.Non-proteolytic Hin-47 analog encapsulated within microparticlesexhibited a more balanced profile with IgG1, IgG2a and IgG2b essentiallyequally represented at either timepoint (day 56 or day 78). For antigendelivered in soluble form or when physically mixed with microparticles,a similar profile was seen by day 78 where significant levels of IgG2acan also be seen.

Thus, via the intranasal route, the TH₁/TH₂ response was essentially thesame irrespective of whether antigen was encapsulated withinmicroparticles, physically mixed with microparticles or in soluble form.

FIG. 10C shows the results for anti-Hin-47 IgA antibody responsesobtained from bleeds on day 78. With soluble antigen no detectable serumIgA was found. However, significant response was seen for antigen thatis encapsulated or physically mixed with microparticles. The highestlevels obtained for antigen either encapsulated or physically mixed withPLGpS microparticles.

The lung lavage conducted on day 78 revealed substantial differenceswith respect to IgG and sIgA detected from secretions. The anti-Hin-47IgG antibody response (FIG. 10D) from soluble antigen was negligible,however antigen encapsulated within microparticles or physically mixedwith PLG microparticles elicited significant levels of IgG from half ofeach group examined. The most reproducible response for IgG was foundwith antigen physically mixed with microparticles derived from PLGpZS orPLGpS where most or all mice examined possessed significant levels ofIgG.

The anti-Hin-47 sIgA antibody response (FIG. 10E) was similar to thatobtained for IgG in FIG. 10D. Soluble antigen response was negligiblewith antigen encapsulated within microparticles or physically mixed withPLG microparticles showing some response. The most significant responsefor sIgA was found with antigen physically mixed with microparticlesderived from PLGpZS or PLGpS where most mice have reasonable levels ofsIgA.

The induction of local protection at mucosal surfaces is oftenassociated with the presence IgG and sIgA in local secretions. Whereasit cannot be ruled out that the intranasal immunization has not alsoresulted in immunization of the upper respiratory tract, it is clearthat for serum IgG antibody response encapsulated or physically mixedantigen with microparticles is effective. To induce local secretions ofIgG and sIgA physically mixing antigen with microparticles and morespecifically microparticles formulated from either PLGpZS or PLGpSappears to be the more suitable method under these conditions.

Example 11

This Example illustrates the immunogenicity of Flu-X-31 or Flu X-31 plusa co-adjuvant BAY R1-005 entrapped in PLG, PLGpZS and PLGpSmicroparticles formed in accordance with the present invention, in miceimmunized subcutaneously.

Groups of six, 6 to 8 week old female BALB/c mice (Charles RiverBreeding Laboratories, Wilmington, Mass.) were immunized subcutaneously(S.C.) with the following amounts of antigen in 250 μL of PBS (pH 7.4)on day 1: PLG, PLGpZS and PLGpS microparticles prepared as described inExample 5 containing 5.0 μg of Flu X-31 (1.5 μg HA) or 5.0 μg of FluX-31 (1.5 μg HA) and 50 μg BAY R1-005 when administered in soluble formor approximately 20 μg BAY R1-005 when co-encapsulated (FIG. 11).

The mice showed no gross pathologies or behavioral changes afterreceiving microparticles that contained encapsulated Flu X-31 or FluX-31 with BAY R1-005. Sera were obtained on days +21 and +33 and wereevaluated for the presence of anti-Flu X-31 IgG antibodies by antigenspecific ELISA. All samples were analyzed in duplicate.

Microtiter plate wells were incubated overnight at room temperature with500 μL of 4.0 μg/mL Flu X-31 in 0.05M carbonate-bicarbonate buffer(pH=9.6). The plates were washed with PBS+0.05% Tween 20 (operationallydefined as washing buffer). Wells were incubated with 200 μL of 5% skimmilk (operationally defined as blocking buffer). After washing withPBS+0.05% Tween 20, the plates were incubated for 1 h at 37° C. with 100μL of sample serially diluted in blocking buffer. Wells were washed withPBS+0.05% Tween 20 and 100 μL of HRP-conjugated antibody (goatanti-mouse IgG (H+L), IgG1, IgG2a or IgG2b) in blocking buffer was addedto each well. After 1 hour incubation at 37° C., the wells were washed5X with PBS+0.05% Tween 20 and 100 μL of freshly prepared colorizingsubstrate [H₂O₂ (9 parts) and TMB (1 part)] is added to each well. After5 minutes incubation in the dark at room temperature the reaction isstopped by adding 50 μL of a 2M H₂SO₄ solution and the optical densityof the fluid in each well was determined at 450 nm using a microplatereader. A normal mouse sera pool was used to establish baseline opticaldensity values in the assay. Hyperimmune mouse Flu X-31 antiserum wasused as a positive control. Pre-immune sera is used as negative control.

The serum antibody titres following immunizations are shown in FIG. 11.The results of a single immunization (day 1) indicated that antigenpresented to the immune system entrapped in PLG, PLGpZS or PLGpSmicroparticles was more immunogenic than soluble antigen alone. The mostrelevant results were obtained with Flu X-31 or Flu X-31 plus BAY R1-005encapsulated within PLGpZS microparticles. Although a sub-optimal doseof BAY R1-005 was encapsulated within all microparticles examined, theimmunogenic response with the PLGpZS microparticles also proved to besignificantly higher than that obtained with soluble Flu X-31 and BAYR1-005 alone.

The studies presented in this Example demonstrate that viral antigensfrom influenza virus can be made more immunogenic and elicit high levelsof serum IgG antibodies, when the antigens are entrapped inmicroparticles formed in accordance with the present invention. Inaddition the significantly higher immunogenicity displayed by themicroparticle systems after a single immunization demonstrates thepotential of these materials for development as single dosage vaccines.

Example 12

This Example illustrates the immunogenicity of Flu-X-31 or Flu X-31 plusa co-adjuvant BAY R1-005 entrapped in PLG, PLGpZS and PLGpSmicroparticles formed in accordance with the present invention, in miceimmunized intranasally.

Groups of six, 6 to 8 week old female BALB/c mice (Charles RiverBreeding Laboratories, Wilmington, Mass.) were immunized intranasally(I.N.) with the following amounts of antigen in 25 μL of PBS (pH 7.4) ondays 1 and 34: PLG, PLGpZS and PLGpS microparticles prepared asdescribed in Example 5 containing 5.0 μg of Flu X-31 (1.5 μg HA) or 5.0μg of Flu X-31 (1.5 μg HA) and 50 μg BAY R1-005 when administered insoluble form or approximately 20 μg BAY R1-005 when co-encapsulated(FIG. 12).

The mice showed no gross pathologies or behavioral changes afterreceiving microparticles that contained encapsulated Flu X-31 or FluX-31 with BAY R1-005. Sera were obtained on days +21, +33 and +92 andwere evaluated for the presence of anti-Flu X-31 IgG antibodies byantigen specific ELISA as described in Example 10. All samples wereanalyzed in duplicate.

Mice immunized I.N. with soluble antigen, soluble antigen plusco-adjuvant or encapsulated antigen showed a similar anti-Flu X-31 IgGantibody response. Interestingly, a reduced (likely due to delayedrelease) immunogenic response for encapsulated antigen plus co-adjuvantrelative to soluble antigen, antigen plus adjuvant or encapsulatedantigen on day +21 and +33 was shown when administered via the nasalroute. However, after the second immunization (day 34) a significantboost in response with these groups was observed resulting inessentially similar immunogenicity on day +92 for all groupsirrespective of adjuvant used.

The results of the I.N. immunizations described in this Example showsthat the immunogenicity of an antigen or antigen plus a co-adjuvantcannot be significantly enhanced by entrapment in microparticles formedin accordance with the present invention.

Example 13

This Example illustrates the immunogenicity of Flu A-Texas or FluA-Texas +BAY R1-005 encapsulated or physically mixed with microparticlesin mice immunized subcutaneously.

It is known that most non-replicating viral vaccines require multipledoses for sufficient serum antibody titres to be protective. Thus, it isstrongly desirable to achieve this by administering a single dose ofantigen.

We sought to examine the immunogenicity of Flu A-Texas or Flu A-Texasplus a co-adjuvant BAY R1-005 which was entrapped in PLGpSmicroparticles or physically mixed with microparticles administered as asingle dose, formed in accordance with the present invention. Groups ofsix, 6 to 8 week old female DBA2 mice (Charles River BreedingLaboratories, Wilmington, Mass.) were immunized subcutaneously (S.C.)with the following amounts of antigen in 250 μL of PBS (pH 7.4) on day1: PLGpS prepared as described in Example 5 containing 1.0 μg of FluA-Texas (0.35 μg HA) or 10.0 μg of Flu A-Texas (3.5 μg HA) or 1.0 μg ofFlu A-Texas (0.35 μg HA) and approximately 2.0 μg of BAY R1-005 or 10.0μg of Flu A-Texas (3.5 μg HA) and approximately 20 μg of BAY R1-005 ormicroparticles prepared as described in Example 5 physically mixed with1.0 μg of Flu A-Texas (0.35 μg HA) and 20 μg of BAY R1-005 or 10.0 μg ofFlu A-Texas (3.5 μg HA) and 20 μg of BAY R1-005 (FIG. 13).

The core loading of PLGpS microparticles containing Flu A-Texas and BAYR1-005 was determined via amino acid analysis (Table 1). The mass ofmicroparticles administered was adjusted such that the required dose ofFlu A-Texas (1.0 μg of Flu A-Texas (0.35 μg HA) for the low dose groupsor 10.0 μg of Flu A-Texas (3.5 μg HA) for the high dose groups) wasdelivered. Thus the dose of BAY R1-005 delivered for the high dosegroups was ten fold greater than that of the low dose groups examined.It is expected that formulation conditions can be adjusted such that thequantity of BAY R1-005 co-encapsulated is comparable.

The mice showed no gross pathologies or behavioral changes afterreceiving microparticles that contained encapsulated Flu A-Texas or FluA-Texas with BAY R1-005. Sera were obtained on days +21 and +42 or +57and were evaluated for the presence of functional antibody via thehemagglutination inhibition antibody assay (HAI). All samples wereanalyzed in duplicate.

The influenza hemagglutination inhibition antibody assay was performedwith heat-inactivated mouse serum that had been incubated for 30 minuteswith 10% chicken red blood cells to remove non-specific inhibitors.Twofold dilutions of sera were added to a 96 well microtiter plate and 8HA units of virus suspension in an equal volume were added to each welland incubated at room temperature for 30 minutes. A 1.0% suspension ofchicken red blood cells were added to each well and incubated at roomtemperature for 30 minutes. Positive and negative reference sera wereincluded in the test to ascertain specificity and sensitivity of thetest. Positive sera from influenza virus immunized animals. Negativesera from PBS immunized animals, titre should be less than or equal to15. The HAI titres are expressed as the reciprocal of the highestdilution that completely inhibits hemagglutination of erythrocytes.

The results of a single immunization (day 1) as shown in FIG. 13indicate that by day +42 antigen presented to the immune system insoluble form or entrapped in PLGpS microparticles (FIG. 13) elicitsnegligible or low functional antibody. For Flu (10 μg) presented to theimmune system as a physical mixture with BAY R1-005 the HAI responseelicited was 8×higher than that obtained with soluble antigen of similardose and 6×higher that that obtained by PLGpS/Flu microparticles ofeither dose. The most relevant results were obtained forPLGpS/Flu(A-Texas)/BAY R1-005 formulated microparticles wherein the HAItitre of the low dose group (1 μg) was 8×that of soluble antigen and thehigh dose group (10 μg) was 32×that of soluble antigen. A moderateincrease in the HAI titre was found for the additional control groupsexamining physical mixtures of Flu with PLGpS microparticles or physicalmixtures of Flu, PLGpS microparticles and BAY R1-005. The HAI responseselicited with physically mixed Flu and PLGpS microparticles was found tobe 4×higher for the low dose group and 8×higher for the high dose groupthan that obtained with soluble antigen of similar dose. The HAIresponses elicited for physical mixtures of Flu with PLGpSmicroparticles and BAY R1-005 was found to be 4×higher than thatobtained with soluble antigen, irrespective of dose.

The studies presented in this Example demonstrate that viral antigensfrom influenza virus elicit high levels of functional antibodies whenthe antigens are entrapped in microparticles in the presence of anadditional adjuvant (as determined by HAI titres), formed in accordancewith the present invention. There is a dose dependence observed and inaddition the significantly higher functional antibody (a correlate forprotection) displayed by the microparticle antigen and adjuvantco-encapsulated systems after a single immunization further demonstratethe potential of these materials for development as single dosage forms.

Example 14

This Example examines the effects of formulation conditions typicallyemployed for microencapsulation on individual components of Flu(trivalent) vaccine.

Flu (trivalent) vaccine contains three homologous HA strains (A/Texas,A/Johannesburg and B/Harbin) in approximately equal amounts. Thequantitation of each specific HA component has been determined by singleradial diffusion assay (SRID) (ref. 32). The SRID assay employspolyclonal sera to detect each HA component and is an effectiveindicator for conformational changes. The minimal concentrationdetectable with this assay for each component is approximately 10 μg/mL.A series of samples containing 2.0 mL of Flu (trivalent) vaccine(concentration=265 μg/mL) were prepared. For each of these samples, theconcentration of A/Texas specific HA=20.25 μg/mL, A/Johannesburgspecific HA=20.60 μg/mL and B/Harbin specific HA=21.42 μg/mL,respectively, was determined by SRID.

The effects of solvents typically employed in microencapsulationprocedures, such as Dichloromethane (DCM) or Ethyl Acetate (EtOAc), wereevaluated under conditions that simulate the homogenization procedure.Short sonication times (30 seconds) were used to model this.Additionally, small amounts of additives, such as BAY (aglyco-lipo-peptide) and DC-Chol (a cationic lipid), were examined todetermine if improved recovery of antigen can be confirmed.

The scale of the reaction was designed to mimic the procedures asdescribed in Example 5. The organic solvent was evaporated from eachmixture prior to SRID analysis and suitable controls were examined todetermine if the organic solvent, homogenizing method or addition of BAYor DC-Chol perturbed the results in any way.

The results of this study are shown in Table 8. The effects ofsonication on the sample were minimal as shown by comparing entries 1and 2. Entries 3 and 4 show that the recovery of antigen was affected bysonication in organic solvents like EtOAc or DCM. When EtOAc isemployed, 75% of A/Texas specific HA and 78% of A/Johannesburg specificHA is recovered. No B/Harbin specific HA component was detectable bythis method after treatment with EtOAc. This result indicates that lessthan 50% of this component was actually recovered, as the lowerdetection limit is about 10 μg/mL. When DCM was employed as solvent, 85%of A/Texas specific HA, 96% of A/Johannesburg specific HA and less than50% of B/Harbin specific HA component were recovered respectively.

Entries 5 and 6 examine the influence of BAY or DC-Chol as additive inthe organic phase with EtOAc as solvent. For this combination, allcomponents were detectable (at levels>50%) with 65% (BAY) or 82%(DC-Chol) A/Texas specific HA recovered, 82% (BAY) or 70% (DC-Chol)B/Harbin specific HA recovered and 87% (BAY) or 88% (DC-Chol)A/Johannesburg specific HA recovered respectively. Entries 7 and 8examine BAY or DC-Chol as an additive in the organic phase with DCM assolvent. The assay indicates that some components were not completelyrecovered or that the assay is effected in some way. Specifically, 91%(BAY) or 92% (DC-Chol) A/Texas specific HA is recovered. Less than 50%B/Harbin specific HA was recovered in either case. The results forA/Johannesburg specific HA are inconclusive due to deviations fromlinearity with these combinations. This occurred only when DCM was usedas solvent.

Maximum recovery of all components was obtained for formulationsemploying Flu (trivalent) vaccine in EtOAc with BAY or DC-Chol asadditive. This Example indicates that materials with lipophilicproperties, such as BAY or DC-Chol, can be used to stabilize vaccineformulations containing sensitive components. Materials with theseproperties can function at interfaces such that proteins are protectedfrom the air/water hydrophobic surface during homogenization. Proteinconformation is particularly sensitive to these effects. The SRID assayresults reported in this Example support these conclusions.

Example 15

This Example illustrates the single dose immunogenicity of Flu(trivalent) or Flu (trivalent)+adjuvant cocktails coencapsulated withinPLGpS microparticles in mice immunized subcutaneously.

Flu (trivalent) vaccine contains three homologous HA strains (A/Texas,A/Johannesburg and B/Harbin) in approximately equal amounts. The totalHA content in 10.0 μg of flu (trivalent) vaccine is 2.35 μg. Eachspecific HA component has been determined by single radial diffusionassay (SRID) (ref. 32) to be 0.76 μg A/Texas, 0.81 μg A/Johannesburg and0.78 μg B/Harbin respectively.

In this study the dose of HA administered for each component strain wassignificantly less than that used in Example 13. For comparison, themonovalent vaccine used in Example 13 contained approximately 3.25 μg ofA/Texas specific HA.

Mice were immunized by the subcutaneous route in the presence ofadjuvants chosen based on Th₂/Th₁ profile; DC-Chol (a cationic lipid)and BAY R1-005 (glyco-lipo-peptide with more balanced Th₂/Th₁), PCPP(poly[di(carboxylatophenoxy)-phosphazene] sodium salt, (Virus ResearchInstitute, Cambridge Mass.), primarily Th₂. PLGpS microparticles havebeen shown to induce a more balanced Th₂/Th₁ profile, as described inExample 8.

Groups of eight 6 to 8 week old female DBA-2 mice (Charles RiverBreeding Laboratories, Wilmington, Mass.) were immunized subcutaneously(S.C.) with the following amounts of antigen in 250 μL of PBS (pH 7.4)on day 1: PLGpS microparticles prepared as described in Example 5containing 10.0 μg of Flu (trivalent−2.35 μHA) or 10.0 μg of Flu(trivalent−2.35 μg HA) and BAY R1-005, DC-Chol or PCPP (Table 5).

The core loading of PLGpS microparticles containing Flu (trivalent) wasdetermined via amino acid analysis (Table 6). Excellent recovery ofantigen was found for all PLGpS microparticle formulations (>70%). Thisresult is consistent with our findings that the initial concentration ofprotein solution can dramatically effect the encapsulation efficiencies.

The mass of microparticles administered was adjusted such that therequired dose of 10.0 μg of Flu (trivalent−2.35 μg total HA) wasdelivered. No attempt to optimize the dose of adjuvant coadministeredwas made, although it is expected that this would be a function offormulation conditions.

The mice showed no gross pathologies or behavioral changes afterimmunization with PLGpS microparticles that contained encapsulated Flu(trivalent) vaccine or Flu (trivalent) vaccine and adjuvants cocktails.Sera were obtained on days 21, 42 and 57 and were evaluated for totalIgG, IgG1, IgG2a and for the presence of functional antibody via thehemagglutination inhibition antibody assay (HAI). Each strain (A/Texas,A/Johannesburg and B/Harbin) was examined to evaluate the effects ofencapsulation and release on immunogenicity and functional antibody in amulti-component system. All samples were analyzed in duplicate. Theantibody ELISA's were performed as described in Example 13.

FIGS. 14a to c, show the specific IgG antibody responses elicited byPLGpS/Flu (trivalent) microparticle formulations, to each straincontained within the Flu (trivalent) vaccine.

By day 57, the highest total IgG titres were found for PLGpS/Flu(trivalent)/DC-Chol microparticle formulations. This result was true foreach strain examined (A/Texas—32×response of soluble Flu control,A/Johannesburg—64×response of soluble Flu control, B/Harbin—64×responseof soluble Flu control).

By day 57, slightly higher IgG antibody responses, as indicated bytitre, to A/Texas (2×response of soluble Flu control) and A/Johannesburg(4×response of soluble Flu control) were found forPLGpS/Flu(trivalent)/PCPP microparticle formulations.

By day 57, essentially identical total specific IgG antibody responses(irrespective of strain), as indicated by titre, were elicited for thePLGpS/Flu (trivalent), PLGpS/Flu (trivalent)/BAY microparticleformulations and the soluble Flu (trivalent) vaccine control group. Thisis consistent with the results obtained in Example 13 with monovalentvaccine where similar results were observed for these three systems.

To determine the influenza hemagglutination inhibition antibody assay(HAI) for each HA strain, serum samples were heated at 56° C. for 30 minto inactivate complement and pre-treated with trypsin (0.1 mL of 8mg/mL)/periodate (12.5 μL-0.001 M) to destroy endogenous inhibitors ofhemagglutination. Serially diluted antisera were tested in duplicate fortheir ability to inhibit the agglutination of 1% chick red blood cellsby 4 HAU of A/Texas, A/Johannesburg or B/Harbin virus respectively in astandard HAI assay (ref. 33).

FIGS. 15a to c, show the specific HAI titres elicited to each HA straincontained within the trivalent vaccine. By day 57, the highestsustainable HAI titres were found for PLGpS microparticles formulatedwith Flu (trivalent) vaccine in the presence of DC-Chol (HAI titre; 240to HA specific A/Texas (8×soluble Flu (trivalent) control), 240 to HAspecific A/Johannesburg (16X soluble Flu (trivalent) control) and 60 toHA specific B/Harbin (8×soluble Flu (trivalent) control)). Notablyhigher titres were also found for PLGpS micropartilcles formulated withFlu (trivalent) in the presence of BAY (HAI titre; 60 to HA specificA/Texas (2×soluble Flu (trivalent) control), 60 to HA specificA/Johannesburg (4×soluble Flu (trivalent) control) and 15 to HA specificB/Harbin (equal to soluble Flu (trivalent) control)). The HAI titresfound for BAY formulations with Flu (trivalent) vaccine, in thisExample, are comparable to those obtained for the low dose groups inExample 13, wherein 1 μg Flu (monovalent) vaccine −0.325 μg A/Texasspecific HA was administered. PLGpS/Flu (trivalent)/PCPP microparticleformulations failed to elicit any detectable functional antibodyresponse.

An experiment was conducted examining a higher dose of PLGpS/Flu(triv)/DC-Chol formulated microparticles. This group of mice wasimmunized on day 1 with approximately twice the dose of Flu (triv)vaccine administered to the other groups (Dose—Flu(triv)=21.2 μg, totalHA=5.00 μg). The results obtained for this high dose are included inFIGS. 15a to c. The HAI titres elicited for a control group immunizedwith soluble Flu (triv) vaccine at this dose were marginal andessentially equivalent to the low dose group examined (results notshown). By day 57, this formulation elicited significantly higherspecific HAI titres than the low dose group (HAI titre; 480 to HAspecific A/Texas (5×soluble Flu (trivalent) control), 480 to HA specificA/Johannesburg (5×soluble Flu (trivalent) control) and 120 to HAspecific B/Harbin (3×soluble Flu (trivalent) control). Additionally, itwas found that the HAI titres elicited were sustainable, beingmaintained at these levels from day 21 through 57.

Additives, such as BAY or DC-Chol, were introduced into the organicphase, containing polymer and organic solvent wherein they can functionat the interface to protect antigen during formulation procedures (seeExample 13, Table 8). This serves to increase the recovery of antigenicmaterial.

In addition, in vitro release studies have shown that a sizable amountof the encapsulated components are released within the first three days.It has been experimentally determined that about 5 to 15% of antigenencapsulated is detectable at this stage of release (antigen which issurface localized on the microparticle). The priming immunization wassignificantly enhanced by the co-release of adjuvants. This is likelythe result of being localized in close proximity to antigen during theinitial release phase.

It is noteworthy that PLGpS microparticle formulations coencapsulatingantigens with adjuvants, such as PCPP, did not result in formulationswith improved vaccine efficacy. This material was added to the aqueousphase of the primary emulsion and does not possess the lipophilicproperties of DC-Chol or BAY. It has been demonstrated that PCPP hasstrong adjuvant properties in other systems, yet in this Example, amarginal improvement in encapsulation efficiency or adjuvancy of themicroparticle formulation is observed.

The studies presented in this Example demonstrate that viral antigensfrom influenza virus in a multi-component system can elicit high levelsof total IgG antibody and functional antibodies (HAI) when the Fluantigens are entrapped in microparticles in the presence of anadditional co-encapsulated adjuvant, such as BAY or DC-Chol.

Example 16

This Example evaluates the infection rate and protection after singledose subcutaneous immunization with PLGpS microparticle formulations inthe mouse model.

A/Taiwan/1/86 (H1N1) as live influenza viruses in egg-derived allantoicfluid, mouse-adapted A/Taiwan/1/86 and commercial A/Taiwan/1/86monovalent subunit vaccine (Fluzone®) were obtained from PasteurMérieux, Connaught, USA (Swiftwater, Pa.).

Groups of 8 female DBA-2 mice aged 6 to 8 weeks (described in Example14) were immunized subcutaneously on day 1 with Flu (trivalent) vaccinecontaining A/Texas, A/Johannesburg and B/Harbin strains (2.35 μg totalHA in 0.25 mL PBS). Control mice received either PBS alone or 400hemagglutination units (HAU) of live A/Texas virus as allantoic fluid.Fourteen days later, mice were challenged intranasally while underanesthesia with 50 μL of live mouse-adapted A/Taiwan/1/86 (5 LD50) inallantoic fluid. Protection was assessed by monitoring mortality dailyand morbidity (weight change) every 2 days up to 14 days post-challenge.

FIG. 16 shows the results of the challenge. As expected, all miceimmunized with homologous live (A/Texas) virus suffered minimal loss inweight and fully recovered within 2 weeks of challenge as measured bythe weight percent change from baseline. Also as expected, miceimmunized with PBS suffered a precipitous drop in weight which by day 10fell below 30% of baseline. No mice in this control group survived after10 days.

Mice immunized with PLGpS/Flu (trivalent)/DC-Chol microparticleformulations were successfully infected, as indicated by the 8% averagedrop in weight within 4 days post challenge. Of the PLGpS formulatedgroups examined, these mice exhibited the swiftest recovery, reaching100% of baseline after 2 weeks. The entire group recovered indicating aprotective titre was raised in each mouse.

Mice immunized with PLGpS/Flu (trivalent)/BAY microparticle formulationswere also successfully infected, as indicated by the 10% average drop inweight by day 10. These mice recovered more slowly, reaching 95% ofbaseline after 2 weeks. The entire group recovered indicating that aprotective titre was raised in all of these mice, although the rate ofrecovery indicates the level of protection was less than that seen forthe DC-Chol group at this dose. This is in accordance with the HAIvalues determined in Example 15 for these two groups.

The soluble Flu (trivalent) vaccine control group and the PLGpS/Flu(trivalent) or PLGpS/Flu (trivalent)/PCPP microparticle formulationswere less successful. These groups experienced a 23 to 29% average dropin weight by days 8-10 post challenge. The rate of recovery was slowestfor these mice reaching 86 to 92% of baseline after 2 weeks.Additionally not all mice in these groups survived. Six of eight micesurvived from the PLGpS/Flu (trivalent) microparticle formulated group.Seven of eight survived from the PLGpS/Flu (trivalent)/PCPP formulatedgroup and six of eight survived from the soluble Flu (trivalent) controlgroup. In each of these cases no or low protection is observed.

In Example 14, we evaluated the effects of microencapsulationformulation conditions on the Flu (trivalent) vaccine. Strain-specific(A/Texas, A/Johannesburg and B/Harbin) analysis of antigen recovery bySRID suggested that minimal loss in antigenic activity for all threestrains in this multi-component system could be expected when employingDC-Chol or BAY in the organic phase with PLGpS polymer and EtOAc assolvent.

In Example 15, the higher functional antibody (a correlate forprotection) elicited by single subcutaneous administration of PLGpS/Flu(trivalent) vaccine formulated in the presence of DC-Chol or BAYdemonstrated the utility of these formulations in the mouse model. Theresults of the challenge/protection study, described in Example 16, arein agreement with the ranking for protection as suggested by thefunctional antibody studies.

The delivery of antigen and adjuvant within a biodegradablemicroparticle can result in more efficient presentation to the immunesystem. The adjuvancy found for soluble mixtures of DC-Chol or BAY withantigen(s) may be significantly increased by employing this formulationstrategy. The possibility for fewer and lower (antigen/adjuvant) doseregimens is indicated by these results. Specifically, this Examplestrongly indicates the potential of these new microparticle basedformulations for development as a single efficacious dosage form.

Example 17

This Example illustrates the immunogenicity of transferrin bindingprotein (Tbp-2) derived from Moraxella catarrhalis (as described in WO97/13785, assigned to the assignee hereof and the disclosure of which isincorporated herein by reference) encapsulated in PLGpS microparticles,Tbp-2 physically mixed with PLGpS microparticles or Tbp-2 formulatedwith Alum in mice immunized subcutaneously.

Groups of five, 6 to 8 week old female BALB/c mice (Charles RiverBreeding Laboratories, Wilmington, Mass.) were immunized subcutaneously(S.C.) with the following amounts of antigen in 250 μL of PBS (pH 7.4)on days 1, 28 and 43: PLGpS microparticles prepared as described inExample 5 containing 0.3 μg of Tbp-2; PLGpS microparticles prepared asdescribed in Example 5 physically mixed with 0.3 μg of Tbp-2; and Alum(1.5 mg/dose) formulated with 0.3 μg of Tbp-2 (Table 5).

The mice showed no gross pathologies or behavioral changes afterreceiving microparticles that contained encapsulated Tbp-2,microparticles physically mixed with Tbp-2 or Alum physically mixed withTbp-2. Sera were collected on days +14, +27, +42 and +55 and wereevaluated for the presence of anti-Tbp-2 IgG antibodies by antigenspecific ELISA as described in Example 8. All samples were analysed induplicate.

The core loading of PLGpS microparticles containing tbp-2 was determinedvia amino acid analysis (4.0 μg/mg (32.8%)) as described in Example 6.

The results of immunizations (FIG. 17a) indicate that antigen presentedto the immune system entrapped in PLGpS microparticles elicited asubstantially higher titre than that obtained for soluble antigen or forantigen physically mixed with PLGpS microparticles alone. In addition,this study indicates that microencapsulated formulations were asimmunogenic as the traditional Alum adjuvanted systems. The kinetics ofthe immune response were found to be similar for both formulations.

The IgG subtype profile (FIG. 17b) for the bleed obtained on day 55revealed differences in the immune responses elicited for Tbp-2encapsulated in PLGpS microparticles and Tbp-2 physically mixed withPLGpS microparticles or formulated with Alum. IgG1 is the dominantsubtype detected (with some IgG2b) when antigen was administered insoluble form or as a physical mixture with PLGpS microparticles orformulated with Alum.

In this Example, the IgG subtypes induced by Tbp-2 encapsulated withinPLGpS microparticles elicit comparatively more IgG2a. This trend is asimilar trend to that observed for Hin-47 in Example 8.

These results suggest that the mechanisms of immune response induced byimmunizing with encapsulated antigens are different from that generatedwith Alum. A more balanced Th₂/Th₁ profile (as indicated by theIgG2a:IgG1 ratio) is exhibited when antigen is administered encapsulatedwithin microparticles.

As may be seen from the results herein, the quality of the immuneresponse mediated by PLGpS microparticles encapsulating antigen issubstantially different from that obtained by physically mixing withPLGpS microparticles, formulating with Alum or by administering solubleantigen alone. Additionally, the magnitude of the immune responseinduced by Alum in antigen/Alum formulations is comparable to thatprovided by microparticles containing encapsulated antigen.

Example 18

This Example illustrates the immunogenicity of Tbp-2 encapsulated inPLGpS microparticles and Tbp-2 physically mixed with PLGpSmicroparticles in mice immunized intranasally.

Groups of five, 6 to 8 week old female BALB/c mice (Charles RiverBreeding Laboratories, Wilmington, Mass.) were immunized intranasally(I.N.) with the following amounts of antigen in either 10 μL or 50 μL ofPBS (pH 7.4) on days 1, 28 and 43: PLGpS microparticles prepared asdescribed in Example 5 containing 6.0 μg of Tbp-2; and PLGpSmicroparticles prepared as described in Example 5 physically mixed with6.0 μg Tbp-2 (Table 5).

The mice showed no gross pathologies or behavioral changes afterimmunization with PLGpS microparticles that contained encapsulated Tbp-2or PLGpS microparticles that were physically mixed with Tbp-2. Sera wereobtained on days 14, 27, 42 and 55 and were evaluated for the presenceof anti-Tbp-2 IgG antibodies by antigen specific ELISA as described inExample 8. All samples were analyzed in duplicate.

The core loading of PLGpS microparticles containing Tbp-2 was determinedvia amino acid analysis (4.0 μg/mg (32.8%)) as described in Example 6.

The serum IgG Tbp-2-specific antibody titres following I.N. immunizationis shown in FIG. 18. When the volume of the dose administered was low(10 μL) these results indicate that an antigen (Tbp-2) incorporated intoor physically mixed with PLGpS microparticles is less immunogenic thansoluble antigen of similar dose (6.0 μg).

When the volume of the dose was increased (50 μL), the overall titresare significantly higher for all groups. These results indicate that anantigen (Tbp-2) physically mixed with PLGpS microparticles issubstantially more immunogenic than microparticle encapsulated antigenor for soluble antigen of similar dose (6.0 μg) in accordance withearlier observations made for intranasal immunization of Hin-47 (Example10).

In our initial study with intranasal Hin-47 immunizations, Example 10,it was found that a strong humoral response and robust secretoryresponse were obtained by administering Hin-47 physically mixed withmicroparticles. This study with Tbp-2 has confirmed the earlierobservations. Additionally, we have established that the volume of thedose administered plays a significant role in the type and strength ofthe immune response invoked.

Example 19

This Example illusrates the release of antigen from the microparticles.

Having established that antigen encapsulated within PLGpS microparticlesis as immunogenic as Alum formulations, we next sought to examinewhether the prime and delayed release character of antigens encapsulatedwithin these polymeric matrices could be exploited so that fewerimmunizations would be needed.

In this Example, we examined the immunogenicity of Tbp-2 encapsulated inPLGpS microparticles , physically mixed with PLGpS microparticles orformulated with Alum or CFA/IFA in guinea pigs immunized subcutaneously.

Groups of two, 6 to 8 week old female guinea pigs (Charles RiverBreeding Laboratories, Wilmington, Mass.) were immunized subcutaneously(S.C.) with the following amounts of antigen: PLGpS microparticlesencapsulating 5.0 μg of tbp-2 suspended in 500 μL of PBS (pH 7.4) ondays 1 and 28, prepared as described in Example 5; Complete FreundsAdjuvant (CFA) formulated in PBS (pH 7.4) with 5.0 μg of Tbp-2 on day 1followed intramuscularly (I.M.) by Incomplete Freunds Adjuvant (IFA)formulated in PBS (pH 7.4) with 5.0 μg of Tbp-2 on days 14 and 28; orAlum formulated in PBS (pH 7.4) with 5.0 μg of Tbp-2 on days 1, 14 and28 (Table 5).

The guinea pigs showed no gross pathologies or behavioral changes afterimmunization with PLGpS microparticles that contained encapsulatedTbp-2, PLGpS microparticles physically mixed with Tbp-2, CFA/IFAformulated Tbp-2 or Alum formulated Tbp-2. Sera were obtained on days 40and 56 and were evaluated for the presence of anti-Tbp-2 IgG antibodiesby antigen specific ELISA. All samples were analyzed in duplicate. Theantibody ELISA's are described in Example 8.

The core loading of PLGpS microparticles containing Tbp-2 was determinedvia amino acid analysis (4.0 μg/mg (32.8%)) as described in Example 6.

The results shown in FIG. 19 indicate that two doses of PLGpSmicroencapsulated Tbp-2 elicited similar anti-Tbp-2 antibody responsesas 3 doses of Tbp-2 formulated either in CFA/IFA or with Alum.

The present results strongly suggest that microencapsulated Tbp-2 may beexploited as a candidate vaccine for two dose schedules.

Example 20

This Example illustrates the immunogenicity of rUrease orrUrease/adjuvant cocktails encapsulated within PLGpS microparticles inmice immunized subcutaneously or intragastrically.

Groups of eight, 6 to 8 week old outbred Swiss female mice (Janvier,France) were immunized subcutaneously (S.C.) with the following amountsof antigen in 300 μL of PBS (pH 7.4) on days 1 and 28 and 56: PLGpSmicroparticles prepared as described in Example 5 containing 10.0 μg ofrUrease; and PLGpS microparticles prepared as described in Example 5coencapsulating 10.0 μg of rUrease and DC-Chol, PCPP or CT-X; 10.0 μg ofrUrease plus soluble DC-Chol (65 μg/dose) or PCPP (100 μg/dose) ascontrols or intragastrically (I.G.) with the following amounts ofantigen in 300 μL of 0.15 M NaHCO₃ (pH 9.0) on days 1, 28 and 56: PLGpSmicroparticles prepared as described in Example 5 containing 40.0 μg ofrUrease; and PLGpS microparticles prepared as described in Example 5coencapsulating 40.0 μg of rUrease and DC-Chol, PCPP or CT-X (Table 5).

The core loading of PLGpS microparticles containing rUrease wasdetermined via amino acid analysis or polyclonal rUrease specific ELISA(Table 7) as described in Example 6. The polyclonal ELISA assayperformed on PLGpS microparticle extracts typically provides a measureof total protein (recovered epitope) encapsulated. Control experimentshave allowed us to quantify the extent to which the solvent/base (SDS)extraction procedure effects the integrity of the antigen. Under theconditions employed we have estimated that about 65 to 75% of theextracted protein remains fully detectable by this assay. Thus thisdetermination will be lower than the measure of total protein recoveredvia amino acid analysis (AAA). Suitable controls were included to ensurethat the presence of the adjuvants (DC-Chol, CT-X or PCPP) did notinterfere with the results obtained. The presence of DC-Chol did notseem to influence the assay, however, PCPP provided anomalous valueswhich were uncharacteristically higher when compared to the totalprotein by AAA in some analyses. In the case of CT-X, a separatepolyclonal ELISA assay was developed to quantify the amount of CT-Xcoencapsulated.

The mass of microparticles administered was adjusted such that therequired dose of 10.0 μg of rUrease (subcutaneously) or 40.0 μg ofrUrease (orally) was delivered.

The mice showed no gross pathologies or behavioral changes afterimmunization with PLGpS microparticles that contained eitherencapsulated rUrease or coencapsulated mixtures of rUrease and adjuvant.Sera were obtained on day 85 and were evaluated for the presence ofanti-rUrease IgG1 and IgG2a antibodies by antigen specific ELISA. Allsamples were analyzed in duplicate.

ELISA's were performed according to standard protocols (biotinylatedconjugates, streptavidine peroxidase complex were from Amersham ando-phenylenediamine dihydrochloride (OPD) substrate from Sigma). Plateswere coated overnight at 4° C. with H. pylori extracts (5 μg/mL) in0.05M carbonate-bicarbonate buffer (pH 9.6). After saturation with BSA(Sigma), plates were incubated with sera (1.5 hrs), biotinylatedconjugate (1.5 hrs), strepavidin peroxidase complex (1 h) and substrate(OPD—10 min). A polyclonal mouse serum directed against H. pyloriextract served as a control in each experiment. The titres wereexpressed as the inverse of the dilution giving 50% of the maximalabsorbance value at 492 nm. Pre-immune sera is used as negative control.

Mice were immunized by the subcutaneous or intragastric route in thepresence of adjuvants chosen based on Th₂/Th₁ profile (DC-Chol—morebalanced Th₂/Th₁, PCPP—primarily Th₂) or for known mucosal adjuvancy(Cholera Toxin (CT-X) or heat labile enterotoxin from E. coli (LT)).PLGpS microparticles have been shown to induce a more balanced Th₂/Th₁profile relevant to other typical adjuvants, such as Alum.

The IgG subtype profile for pooled bleeds obtained on day 85 aftersubcutaneous immunizations is shown in FIG. 20. In all cases, the IgG1response was higher than the IgG2a response (IgG2a:IgG1 ratio rangesfrom 0.08 to 0.33) when PLGpS/rUrease microparticle formulations wereadministered subcutaneously. The total IgG1 response for the solublecontrol groups (rurease+DC-Chol or PCPP) was in the same absolute range.There are differences noted for the IgG2a responses between adjuvantsystems that were either coencapsulated with antigen or administered asa physical mixture with antigen.

For PLGpS/rUrease microparticles (IgG2a:IgG1=0.33), PLGpS/rUrease/CT-X(IgG2a:IgG1=0.30) and PLGpS/rUrease/DC-Chol (IgG2a:IgG1=0.20), a morebalanced IgG2a:IgG1 antibody response (indicative of Th₂/Th₁ ratio) wasobtained. For comparison, the rUrease+DC-Chol control groupIgG2a:IgG1=0.03.

For PLGpS/rUrease/PCPP microparticle formulations, primarily a Th₂response is noted IgG2a:IgG1 ratio=0.08. The analogous soluble mixtureof rUrease+PCPP is strongly Th₂ biased with a very low IgG2a:IgG1ratio=0.003.

In general, coencapsulation of antigen in the presence of theseadjuvants tends to shift the typical immunological profile towards amore balanced Th₂/Th₁ response.

Immunization via the intragastric route in most cases did not elicit anydetectable systemic response. Notable exceptions to this are thepositive control with LT having a modest systemic response (IgG2a:IgG1ratio=0.1) and for rUrease/PLGpS microparticles coencapsulating CT-X(IgG2a:IgG1 ratio=2.1). Interestingly, for oral immunizations,encapsulated antigen plus mucosal adjuvant induces a strikinglydifferent systemic antibody response. The ratio of IgG2a:IgG1 is nowstrongly in favor of IgG2a indicative of the Th₁ path. Examination ofthe literature reveals that CT-X co-administered with antigen orallytypically induces an immune response similar to that elicited by LT.Primarily an IgG1 or TH₂ type of response is reported (ref. 34). Thisstudy illustrates that the quality of the immune response may be changedas a consequence of coencapsulation in PLGpS microparticles.

Example 21

This Example evaluates the infection rate and protection aftersubcutaneous or oral immunizations with PLGpS microparticle/rUreaseformulations in the mouse model.

Mice were challenged 6 weeks after the 2′nd boost (Day 85) by gastricgavage with 300 μL of a suspension of H. pylori bacteria (3×10⁶ cfu).Based on in vitro release studies (Example 7), antigen release frommicroparticles requires approximately 4 weeks, thus the challenge wasscheduled for 2 weeks after this time point.

Four weeks after the challenge, mice were killed and stomachs weresampled to evaluate urease activity (Jatrox test, Procter and Gamble) ina sterile flow hood. Urease activity was assessed 24 hours post-mortemby measuring the absorbance at 550 nm. The principle of the test is thatthe urea present in the test medium is split by Helicobacter pylonurease. The rise in pH causes a color change in the indicator which islikewise present in the test medium (phenol red), from yellow to pinkred.

Mice were infected with a streptomycin-resistant mouse-adapted strain(X43-2AN) of H. pylori. The infection rate was reproducible; 100% of themice were infected in all experiments, as judged by urease activitymeasured in the stomach (Jatrox test). It is to be noted that thestreptomycin resistance of the challenge strain allows it to grow on ahighly selective medium, making this test very sensitive (nocontaminants coming from the normal flora).

This strain was stored at −70° C. in Brucella Broth (BB) (Biomerieux)supplemented with 20% v/v glycerol and 10% v/v foetal bovine serum (FBS)(Hyclone). The challenge suspension was prepared as follows: forpre-culture, H. pylon was grown on Mueller-Hinton Agar (MHA; Difco)containing 5% v/v sheep blood (Biomerieux) and antibiotics:Thrimethoprim (5 μg/mL), Vancomycin (10 μg/mL), Polymixin B sulphate (5μg/mL), Amphotericin (5 μg/mL) and Streptomycin (50 μg/mL) selectivemarker of H. pylori strain X43-2AN (TVPAS) All antibiotics werepurchased from Sigma. MHA-TVPAS plates were incubated for 3 days at 37°C. under micro-aerobic conditions. The pre-culture was used toinnoculate a 75 cm² vented flask (Costar) containing 50 mL of BBsupplemented with 5% v/v FBS and all antibiotics (TVPAS). The flask waskept under micro-aerobic conditions with gentle shaking for 24 hrs. Thesuspension was characterized by Gram's staining, urease activity (Ureaindole medium, Diagnostic Pasteur), catalase (H₂O₂, 3% v/v) and oxidaseactivity (Biomerieux discs). Viability and motility were checked byphase contrast microscopy. The suspension was diluted in BB to O.D. 550nm=0.1 (which was equivalent to 1×10⁷ CFU/mL).

In this study, mice for which an OD.<0.1 was obtained were consideredprotected or at least have less than 10³ to 10⁴ bacteria/stomach(compared to 10⁵ to 10⁶ in controls). Groups of mice representingunimmunized/infected and unimmunized/uninfected controls were alsoincluded in the study (results not shown).

FIG. 21a, shows that protection (via the subcutaneous route), as judgedby the assay described above, follows the ranking PLGpS/rUrease/DC-Chol(geometric mean=0.092)>PLGpS/rUrease/CT-X (geometric mean=0.105),PLGpS/rUrease (geometric mean=0.163) and PLGpS/rUrease/PCPP (geometricmean=0.269). The LT +rUrease (oral) positive control group (geometricmean=0.136) is the standard for protection in this study. Interestingly,typical results with rUrease plus soluble DC-Chol (geometric mean=0.600)or rUrease plus PCPP (geometric mean=1.07) control groups clearly showthe advantages of coencapsulating antigen and adjuvant as a PLGpSmicroparticle formulation.

Mann-Whitney statistical analysis of the data presents the followingconclusions. The PLGpS/rUrease/DC-Chol and PLGpS/rUrease/CT-Xformulations were not significantly different from the LT+rUreasepositive control group. The PLGpS/rUrease/PCPP, rUrease+DC-Chol andrUrease+PCPP groups were significantly different from these groups(p<0.01) and the PLGpS/rUrease group was significantly different fromthe PLGpS/rUrease/PCPP, rUrease+DC-Chol and the rUrease+PCPP (p<0.01)groups.

From this analysis, the PLGpS/rUrease/DC-Chol (7/8 mice have OD. <0.1)and PLGpS/Urease/CT-X (5/8 mice have OD. <0.1) microparticle groupsexhibited solid protection whereas the PLGpS/rUrease (2/8 mice have OD.<0.1) microparticle group and the rUrease+DC-Chol group (2/10 OD. <0.1)exhibited moderate protection and the PLGpS/rUrease/PCPP (0/8 mice haveOD. <0.1) microparticle group and the rUrease+PCPP groups (0/10 OD.<0.1) exhibited low or no protection respectively. This ranking tends tofollow the more balanced Th₂/Th₁ ratios as determined fromimmunogenicity studies (Example 20).

FIG. 21b shows that no full protection via the oral route was observedfor all groups of mice immunized by PLGpS microparticles containingadditional adjuvants.

Statistical ranking follows the order PLGpS/rUrease/CT-X (geometricmean=0.229) >PLGpS/rUrease/DC-Chol (geometric mean=0.403), PLGpS/rUrease(geometric mean=0.475) and PLGpS/rUrease/PCPP (geometric mean=0.493).The LT+rUrease (oral) group (geometirc mean=0.136) is the positivecontrol for this study.

Mann-Whitney statistical analysis of the data presents the followingconclusions. The LT+rUrease positive control group was significantlydifferent than the PLGpS/rUrease/DC-Chol and PLGpS/rUrease/CT-Xformulations (p <0.01). The PLGpS/rUrease/DC-Chol and PLGpS/rUrease/CT-Xformulations were not significantly different from each other. ThePLGpS/rUrease and the PLGpS/rUrease/PCPP formulations were notsignificantly different form each other.

In this study, the PLGpS/rUrease/CT-X (2/8 mice have OD. <0.1) and thePLGpS/rUrease/DC-Chol (1/8 mice have OD. <0.1) microparticleformulations exhibited partial protection. Comparatively, the positivecontrol group of rUrease plus LT exhibited solid protection (7/8 micehave OD. <0.1).

It was suggested, in Examples 14 to 16, that the presentation ofadjuvant to the immune system by association with delivery vehicles,such as PLGpS, microparticles increases the adjuvants effectivenessresulting in a more efficacious vaccine.

Notably, typical control studies in this Example have examinedimmunogenicity and protection after immunizations with soluble mixturesof adjuvants, such as DC-Chol or PCPP, and rUrease by the subcutaneousroute, and have found that protection was low to moderate. These resultsprovide additional evidence that the presentation of antigen andadjuvant in the form of a particulated matrix can result insubstantially more efficacious vaccines.

Additional benefits, such as reduced adjuvant toxicity and thepossibility of modulating the quality of the immune response which ischaracteristic for the adjuvant employed, have been demonstrated in thisExample. It was experimentally determined that the IgG subtype antibodyresponses to rUrease obtained after oral immunization withPLGpS/rUrease/CT-X formulated microparticles was uncharacteristically infavor of IgG2a (Thipath).

In the case of coencapsulated antigen and CT-X (a known mucosaladjuvant) administered subcutaneously or orally, it is also likely thatthis material stabilizes the antigen within the polymeric matrix duringformulation, storage and release by a mechanism similar to that observedfor microparticles prepared in the presence of HSA or BSA (ref. 35).

In conclusion, this study demonstrates the feasibility and efficacy ofPLGpS microparticle based systemic immunization to induce significantprotection against H. pylori infection in the mouse model. This studyalso demonstrates the feasibility for PLGpS microparticle based oralimmunization to induce partial protection against H. pylori infection inthe mouse model.

Additionally, co-encapsulation of rUrease in the presence of additionaladjuvants (specifically DC-Chol or CT-X), can result in notableimprovement in vaccine efficacy.

SUMMARY OF THE DISCLOSURE

In summary of this disclosure, the present invention provides aparticulate carrier for an agent, particularly one having biologicalactivity, comprising a matrix of polymer and biologically activematerial. The particulate carriers in the form of microparticles areable to efficiently deliver agents to the cells of the immune system ofa subject following mucosal or parenteral administration to produce animmune response. Modifications are possible within the scope of thisinvention.

TABLE 1 Antigen(s) entrapped in PLG, PLGpZS or PLGpS microparticlesAntigen Preparation Hin-47 (1.55 mg/mL) + 800 μL of Hin-47 in aqueousBAY (5.0 mg/mL) internal phase plus 40.0 mg of BAY in the organic phaserD-15 (2.05 mg/mL) 800 μL of rD-15. Hin-47 (1.95 mg/mL) + 400 μL ofHin-47 plus 400 μL rD-15 (1.95 mg/mL) of rD-15. Flu X-31 (2.0 mg/mL) 800μL of Flu X-31 or 800 μL Flu A-Texas (1.48 mg/mL) of Flu A-Texas Flu(2.0 mg/mL) + 800 μL of Flu X-31 or 800 μL BAY (5.0 mg/mL) of FluA-Texas in aqueous Flu A-Texas (1.48 mg/mL) + internal phase plus 40.0mg of BAY (5.0 mg/mL) BAY in the organic phase

TABLE 2 Summary of Microparticle Core Loading and EncapsulationEfficiencies (Hin-47, Hin-47 + Bay R1-005) Epitope Equiv. Protein HIN-47Total Protein Epitope vs Total Adjuvant (Conc.) via ELISA¹ via AAA²Total Protein via AAA² (Adjuvants) Polymer (Encaps. Eff.)³ Encaps.Eff.)³ % (Encaps. Eff.)³ Hin-47 PLG 1.4 ug/mg 2.8 ug/mg 50% (1.55 mg/mL)(10.2%) (20.4%) PLGpZS 5.3 ug/mg 7.5 ug/mg 71% 30.8% (43.5%) PLGpS 1.6ug/mg 3.3 ug/mg 48% (11.6%) (23.9%) Hin-47 PLG 2.5 ug/mg 3.8 ug/mg 66%13.5 (3.4%) (1.55 mg/mL) (20.1%) (30.6%) Bay-R1005 PLGpZS 4.1 ug/mg 5.5ug/mg 75% 15.6 (3.9%) (5.0 mg/mL) (19.8%) (26.6%) PLGpS 6.1 ug/mg 9.2ug/mg 66% 23.2 (4.6%) (39.4%) (59.4%) ¹ELISA values are averages of 2independent measurements of protein obtained after complete hydrolysisof microparticles. ²AAA values are averages of 2 independentmeasurements on protein (or adjuvant) encapsulated within microparticlesand 2 independent measurements on protein (or adjuvant) obtained aftercomplete hydrolysis of microparticles. ³Encapsulation efficiency(Encaps. Eff.) calculated as follows: total mass of protein (oradjuvant) recovered/total amount of protein (or adjuvant) used × 100%

TABLE 3 Summary of Microparticle Core Loading and EncapsulationEfficiencies (rD-15, rD-15 + Hin-47) Epitope Equivalent HIN-47 TotalProtein Protein via ELISA¹ via AAA² (Conc.) Polymer (Encaps. Eff.)³Encaps. Eff.)³ rD-15 PLG 7.8 ug/mg (1.95 mg/mL) (43.7%) PLGpZS 7.1 ug/mg(39.2%) PLGpS 7.3 ug/mg (43.7%) rD-15 PLG 1.6 ug/mg  7.0 ug/mg (1.95mg/mL) +  (8.0%) (35.1%) Hin-47 PLGpZS 5.5 ug/mg 13.1 ug/mg (1.95 mg/mL)(26.1%) (62.1%) PLGpS 2.4 ug/mg (11.6 ug/mg (10.8%) (52.1%) ¹ELISAvalues are averages of 2 independent measurements of protein obtainedafter complete hydrolysis of microparticles. ²AAA values are averages of2 independent measurements on protein encapsulated within microparticlesand 2 independent measurements on protein obtained after completehydrolysis of microparticles. ³Encapsulation efficiency (Encaps. Eff.)calculated as follows: total mass of protein recovered/total amount ofprotein used × 100%

TABLE 4 Summary of Microparticle Core Loadings and EncapsulationEfficiencies for Flu X-31 + Bay R1-005 and Flu A-Texas, Flu A-Texas +Bay R1-005 Protein Total Protein Total Adjuvant (Conc.) via AAA¹ viaAAA¹ (Adjuvants) Polymer (Encaps. Eff.)² (Encaps. Eff.)² Flu X-31 PLG4.7 ug/mg (2.0 mg/mL) (26.1%) PLGpZS 6.1 ug/mg (34.1%) PLGpS 7.1 ug/mg(39.7%) Flu X-31 PLG 5.6 ug/mg 22.9 ug/mg (2.0 mg/mL (35.1%) (5.2%) BayR1005 PLGpZS 8.4 ug/mg 28.1 ug/mg (5.0 mg/mL) (52.5%) (10.0%)  PLGpS 5.0ug/mg 27.4 ug/mg (31.0%) (8.4%) Flu-A-Texas PLGpS 2.7 ug/mg (1.48 mg/mL)(22.8%) Flu A-Texas PLGpS 3.4 ug/mg 11.5 ug/mg (1.48 mg/mL) (31.6%)(3.5%) Bay-R1005 (5.0 mg/mL) ¹AAA values are averages of protein (oradjuvant) isolated after complete hydrolysis of microparticles andprotein (or adjuvant) encapsulated within microparticles. ²Encapsulationefficiency (Encaps. Eff.) calculated as follows: total mass of protein(or adjuvant) recovered/total amount of protein (or adjuvant) used ×100%

TABLE 5 Summary of Microparticle Formulation ProceduresAntigen(s)/Adjuvants entrapped in PLGpS microparticles AntigenPreparation tbp-2 (2.88 mg/ml) 800 μl tbp-2 (diluted to 1.44 mg/ml) inaqueous phase with PBS Flu (Trivalent) (0.533 mg/ml) 800 μL Flu(trivalent) in aqueous internal phase Flu (Trivalent) (0.533 mg/ml) +800 μL Flu (trivalent) in aqueous BAY (3.3 mg/ml) internal phase 40.0 mgof BAY in the organic phase Flu (Trivalent) (0.533 mg/ml) + 800 μL Flu(trivalent) in aqueous DC-Chol (3.3 mg/ml) internal phase 40.0 mg ofDC-Chol in the organic phase Flu (Trivalent) (0.533 mg/ml) + 800 μL ofcombined solution in aqueous PCPP (1.25 mg/ml) internal phase rUrease(1.00 mg/ml) 800 μL rUrease in aqueous internal phase rUrease (1.00mg/ml) + 800 μL of combined solution in aqueous CT-X (0.5 mg/ml)internal phase rUrease (1.00 mg/ml) + 800 μL rUrease in aqueous internalDC-Chol (3.3 mg/ml) phase 40.0 mg of DC-Chol in the organic phaserUrease (1.00 mg/ml) + 800 μL of combined solution in PCPP (1.25 mg/ml)aqueous internal phase

TABLE 6 Summary of Microparticle Core Loadings and EncapsulationEfficiencies for Flu (trivalent) and Flu (trivalent) + Adjuvants ProteinTotal Protein (Conc.) via AAA¹ (Adjuvants) Polymer (EncapsulationEfficiency)² Flu (trivalent) PLGpS 5.0 ug/mg (93.3%) (0.533 mg/mL) Flu(trivalent) (0.533 mg/mL) PLGpS  5.8 ug/mg (104.6%) Bay-R1005 (3.3mg/mL) Flu (trivalent) (0.533 mg/mL) PLGpS 3.9 ug/mg (72.7%) DC-Chol(3.3 mg/mL) Flu (trivalent) (0.533 mg/mL) PLGpS 4.6 ug/mg (86.6%) PCPP(1.25 mg/mL) ¹AAA values are averages of protein isolated after completehydrolysis of microparticles and protein encapsulated withinmicroparticles. ²Encapsulation efficiency calculated as follows: totalmass of protein recovered/total amount of protein used × 100%

TABLE 7 Summary of Microparticle Core Loadings and EncapsulationEfficiencies for rUrease and rUrease + Adjuvants Protein Total ProteinTotal Protein (Conc.) via AAA¹ via ELISA¹ (Adjuvants) Polymer (Encaps.Eff.)³ (Encaps. Eff.)³ rUrease PLGpS 3.44 ug/mg 2.5 ug/mg (1.0 mg/mL)(45.8%) (33.2%) rUrease PLGpS 3.15 ug/mg 2.2 ug/mg (1.0 mg/mL) (44.0%)(30.3%) DC-Chol (3.3 mg/mL) rUrease PLGpS 7.35 ug/mg rUrease = 4.5 ug/mg(1.0 mg/mL) (N/D %)⁴ (53.0%) CT-X CT-X = 2.5 ug/mg (0.5 mg/mL) (59.0%)rUrease PLGpS 5.97 ug/mg 3.3 ug/mg (1.0 mg/mL) (67.2%)  (37.1%)⁵ PCPP(1.25 mg/mL) ¹AAA values are averages of two independent measurements ofprotein encapsulated within microparticles, as described in Example 6.²polyclonal ELISA (capture sandwich antigen assay) conducted onmicroparticle hydrosylates, as described in Example 6. Average of 4determinations. ³Encapsulation efficiency (Encaps. Eff.) calculated asfollows: total mass of protein recovered/total amount of protein used ×100% ⁴N/D = not determinable by AAA as total protein is actually acombination of rUrease and CT-X. ⁵ELISA determination in thc presence ofPCPP was highly variable.

TABLE 8 Flu (trivalent) Formulations Examined by SRID's SRID Flu Samplesconc. = Solvent/Solution Sonicate Additives A/Texas:B/Harbin:A/Johannes265 ug/mL examined (30 sec) (quantity) (ug/mL) Entry #1 none no —20.25:20.6:21.42 Entry #2 none yes — 21.73:19.8:23.05 Entry #3 EtOAc yes— 15.21:N/D:16.73 Entry #4 DCM yes — 17.28:N/D:20.54 Entry #5 EtOAc yesBAY 13.04:16.8:18.63 (20 μg) Entry #6 EtOAc yes DC-Chol 16.54:14.4:18.79(20 μg) Entry #7 DCM yes BAY 18.44:N/D:N/L (20 μg) Entry #8 DCM yesDC-Chol 18.55:N/D:N/L (20 μg) N/D = not determined (below detectionlimit of ˜10 μg/mL). N/L = test failed or was not reproducible underexperimental conditions.

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What we claim is:
 1. A process for making a biodegradable, biocompatiblepolyester, which comprises co-polymerizing at least one α-hydroxy acidmonomer having the formula R₁ R₂COHCO₂H, wherein the R₁ and R₂ groupsare H, linear or branched alkyl units, the alkyl units being representedby the formula C_(n)H_(2n+), and at least one pseudo-α-amino acidmonomer of the formula R₅CHNHR₆CO₂H, wherein the R₅ group is a hydroxymethyl or methyl thiol group and R₆ is an amine protecting group.
 2. Theprocess of claim 1, which comprises: forming a mixture of monomerscomprising said at least one α-hydroxy acid and said at least onepseudo-α-amino acid with an organic solvent solution of anesterification catalyst under inert atmospheric conditions;copolymerizing said monomers in said organic solvent solution; andisolating the resultant polymer from said organic solvent solution. 3.The process of claim 1, wherein the α-hydroxy acids comprise a mixtureof α-hydroxy acids, one of said mixture of α-hydroxy acids having R₁ andR₂ groups which are hydrogen and the other of said mixture of α-hydroxyacids having an R₁ group which is CH₃ and R₂ group which is H.
 4. Theprocess of claim 1, wherein the amine protecting group is selected fromthe group consisting of carbobenzyloxy (CBZ or Z), benzyl (Bn),para-methoxybenzyl (MeOBn), benzyloxymethoxy (BOM),tert-butyloxycarbonyl (t-BOC) and [9-fluorenylmethyl oxy]carbonyl(FMOC).
 5. The process of claim 1, wherein the at least one α-hydroxyacid is selected from the group consisting of L-lactic acid, D,L-lacticacid, glycolic acid, hydroxy valeric acid and hydroxybutyric acid. 6.The process of claim 1, wherein the at least one pseudo-α-amino acid isderived from serine.
 7. The process of claim 1, wherein said at leastone α-hydroxy acid monomer and at least one pseudo-α-amino acid monomerare selected to result in poly-D,L-lactide-co-glycolide-co-pseudo-Z-serine ester (PLGpZS).
 8. The processof claim 1, wherein said at least one α-hydroxy acid monomer and atleast one pseudo-α-amino acid monomer are selected to result in poly-D,L-lactide-co-glycolide-co-pseudo-serine ester(PLGpS).
 9. The process ofclaim 2, wherein said polymer has a molecular weight of about 5000 toabout 40,000 dalton.
 10. The process of claim 2, wherein the polymerformed is deprotected by solid phase catalytic reduction or acidcatalysis.
 11. The process of claim 10, wherein said deprotection is byacid catalysis in the presence of hydrogen bromide in acetic acidsolution.
 12. The process of claim 2, wherein said catalyst is stannous2-ethylhexanoate.
 13. The process of claim 2, wherein saidpolymerization is carried out at a temperature of about 120° C. forabout 28 hours.
 14. The process of claim 2, wherein said organic solventis anhydrous chloroform.
 15. The process of claim 2, wherein saidprocess further comprises forming the polymer into a film.
 16. Theprocess. of claim 2, wherein said process further comprises forming thepolymer into microparticles.